Of Campanian descent, Zambrotta began his professional career with his hometown club Como at age 17 in 1994, and remained with the club for three seasons. In his first season in Serie B, he played only one match. In 1995, however, Como were demoted to Serie C1 and Zambrotta started to play more regularly for the first team, as a winger or as a wide-midfielder. In the 1995–96 and 1996–97 seasons, he made 47 appearances and scored 6 goals, playing in Serie B and Serie C1.

In 1997, Zambrotta’s Serie A career began when his great potential was spotted by Bari boss Eugenio Fascetti. Zambrotta was snapped up by Fascetti after Bari had won promotion to Serie A. He made his Serie A debut on 31 August 1997 in a 2–0 home defeat to Parma, and in his first year in the top flight, he played on the left wing and scored 2 goals in 27 appearances. In the 1998–99 season, following the departure of Nicola Ventola, Zambrotta exploded on to the scene. In matchday one, he scored the only goal of the match against Venezia after just nine minutes into the first half. Zambrotta continued to shine and was the key figure for Bari’s surprise start to the campaign, demonstrating his technical attributes and tactical intelligence and versatility. On matchday seven, he scored the opening goal at the San Siro and helped Bari to win the game 3–2 against Internazionale.

Soon after Zambrotta was brought to Serie A, he gained the attention of the Italian national side. He was selected to play for the Italy under-21 national team for the first time on 25 March 1998 against Malta. In his second game for the U21, against Wales on 22 April 1998, he scored the second goal for Italy on the 43rd minute and helped Italy to win 2–1. In 1998, he played in all six games of the Italy U21 while they won five of them and drew the last one against Spain.

On 10 February 1999, at age 22, the Italian under-21 international was rewarded by the coach of the senior team, Dino Zoff, for his displays. He earned his first cap in a 0–0 friendly against Norway in Pisa, Italy. He became the first player of Bari to play for the national side in 50 years. Throughout the 1998–99 season, he made 32 appearances and scored four goals. Halfway through this season, he was brought to Juventus, by Carlo Ancelotti for reported 30 billion Italian lire (€15,493,707, but 3 billion lire paid via Simone Perrotta’s 50% rights) and would play in the black-and-white jersey starting the season after.

After the transfer to Juventus on 1 July 1999, Zambrotta continued his upward momentum under manager Carlo Ancelotti. He made 32 league appearances with one goal in the first season he played for Juventus. On 14 May 2000, the last match day of the season, Juventus missed out on the 1999–2000 Scudetto, as they were defeated 1–0 away at Perugia in the heavy rain, while Lazio got the three points at home by beating Reggina 3–0, and overcame Juventus by a single point. Zambrotta was brought on in the second half in that game and was later given a red card by the referee, Pierluigi Collina. In his second season for Juventus, 2000–01, Zambrotta made 29 league appearances and scored three goals, but Juventus finished once again in second place in Serie A, behind Roma. Zambrotta won his first Scudetto in the 2001–02 season, while he made 32 league appearances and scored one goal during the season, also reaching the 2002 Coppa Italia Final with the club. After Zambrotta suffered an injury in the game against South Korea during the 2002 World Cup, he missed the beginning of the 2002–03 Serie A campaign. During his absence, the new signing of Mauro Camoranesi took his place on the right wing and was in superb form. This prompted then coach, Marcello Lippi, to switch Zambrotta to the left-back when he returned to action. Zambrotta adapted to his new position very quickly and performed particularly well, demonstrating his pace, stamina, work-rate, technical ability, his tactical intelligence and versatility, as well as his ability to contribute both offensively and defensively.

It was on tour with the under-21 team that Zambrotta earned the nickname “the Mermaid” for his allegedly spending one hour every night in front of the mirror grooming at his now trademark goatee. After some impressive displays for the U21 and an excellent start to the 1998–99 season, Zambrotta earned his first senior cap on 10 February 1999, nine days before his 22nd birthday, in an 0–0 friendly draw with Norway.

In 2003, after the retirement of Paolo Maldini from the national team and a successful switch to left-back with Juventus, Zambrotta became the first choice for this position in the national team. On 30 May 2004, Zambrotta scored his first ever international goal in a warm-up against Tunisia. A Bernardo Corradi header was flapped out for Zambrotta’s fierce volley into the roof of the net in the 89th minute while Italy won the game by 4–0. In Euro 2004, although the performance of Italy was largely disappointing and the team was eliminated at the group stage, Zambrotta was one of the few Italian players to be praised for his performances, and was elected to be part of the Team of the Tournament. In Italy’s final group match against Bulgaria, which ended in a 2–1 victory the Italians, he was involved in both of his team’s goals, setting up Antonio Cassano’s injury time winner, although Italy finished third in their group on direct encounters, following a three-way five point tie with Sweden and Denmark.

Zambrotta remained a regular first team player as Juventus won their second consecutive Scudetto and reached the Champions League final, in which they were defeated by Milan in a penalty shoot-out after a goalless draw. The following season, Juventus avenged the defeat by beating Milan to the 2003 Supercoppa Italiana on penalties, and also reached the 2004 Coppa Italia Final, although they failed to retain their league title or progress in Europe. Zambrotta won four Scudetti with Juventus, making a total of 217 league appearances and scoring seven goals in six seasons. In the 2005–06 season, following the injury of Jonathan Zebina, as well as the arrival of Giorgio Chiellini and his successful switch with the national team, Zambrotta was again switched to the right-back. Since then, Zambrotta switched between right and left-fullback or wingback positions frequently throughout the season. In 2005, Zambrotta extended his contract to 2010 but, following Juventus’ relegation to Serie B due to the 2006 Italian football scandal (calciopoli), as well as the revocation of their 2004–05 and 2005–06 Serie A titles under Fabio Capello, he decided to leave the club in the summer of 2006, with Milan, Chelsea, Real Madrid and Barcelona widely tipped to sign him. He was eventually transferred to Barcelona for €14 million.

As of 2005, Zambrotta is married to Valentina Liguori, an Italian model; they announced the birth of their first son, Riccardo, in August 2012.

On 4 August 2006, after his holiday following the 2006 World Cup, Zambrotta reported to his new club Barcelona for the first time in Los Angeles, where Barça were staying for their pre-season tour in North America. He trained with his new teammates and made his debut in the final match of the tour, on 12 August against the New York Red Bulls. Zambrotta’s official debut in Barça shirt was on 17 August against local rivals Espanyol in the first leg of the Supercopa de España. On 17 March 2007, he scored his first goal in the league game against Recreativo de Huelva. He also scored goals in matches against Atlético Madrid at the Vicente Calderón Stadium (Atlético’s record largest home loss, 0–6) and Gimnàstic de Tarragona. By his own admission, his performance at Barcelona had been below-par. He is well remembered by his performance against Manchester United in the semi-final return clash – he marked perfectly the future Ballon d’Or winner Cristiano Ronaldo all game, but made a poor clearance in midfield that ultimately led to Paul Scholes’ long-range effort that decided the match and the two-legged tie itself. In two seasons with the club, Zambrotta made 58 league appearances, scoring three goals, before his return to Italy. He was heavily tipped to join Milan or return to former club Juventus.

New coach Marcello Lippi, who had just resigned as the Juventus manager where he had managed Zambrotta, moved him to right-back. He became a core player in the 2006 World Cup qualifying campaign, featuring in eight of the ten matches to help Italy qualify as group winners. On 15 May 2006, he was chosen as one of the 23 players to participate in the finals in Germany.

During final preparations, Zambrotta injured his left thigh during a training match at the national team’s training base at Coverciano on 28 May 2006. He picked up the injury in a training match against amateur team San Giminiano. This brought certain fears for the Italian squad and Lippi decided to keep Daniele Bonera on stand-by in case Zambrotta could not recover in time. Medical ultrasonography scans on Zambrotta’s thigh in the final days before Italy’s first group match revealed that he would only miss the opening game against Ghana.

Zambrotta was named honorary president of his hometown club Como in 2007.

On 31 May 2008, Zambrotta signed a three-year contract with Milan. The club paid €9 million to Barcelona, with a potential €2 million added on depending on Milan’s performance in the Champions League, for his services. Zambrotta would reportedly be earning €4 million per year. His move forced Milan right back Massimo Oddo to be loaned out to Bayern Munich for more chance to play regularly.

On 21 September 2008, Zambrotta scored his first goal for Milan, from 31 metres out, in the league game against Lazio. In the 2008–09 season, Zambrotta played more games than any other Milan player. In the 2009–10 season, Zambrotta mainly played at left back, competing with Luca Antonini for a starting spot, while the emerged star Ignazio Abate consolidated the right back position as his own.

Late in 2008, World Cup-winning coach Marcello Lippi returned to coach of Italy, selecting Zambrotta for the 2009 FIFA Confederations Cup. Italy, however, failed to progress past the first round, eliminated on goal differential after finishing level on points with the United States.

Zambrotta was selected for the 2010 World Cup by Lippi. He made his first appearance as Italy’s captain in a 1–1 away draw against Switzerland in a pre-World Cup friendly, on 5 June 2010. Despite the Italian team’s struggles throughout the tournament, Zambrotta was one of his nation’s strongest performers in the group stage. Italy earned 1–1 draws in their first two games against Paraguay and New Zealand, collecting two points, and subsequently needing a draw in their final group match against Slovakia in order to proceed the second round. The team’s final fixture against Slovakia saw two Italian goals being disallowed by the referee, as Italy lost the match 3–2, and were eliminated from the tournament in the first round.

After the World Cup, Lippi resigned, while Cesare Prandelli was named as his replacement. In the first matches under Prandelli, Zambrotta was largely omitted from the squad, but he returned to play in several Euro 2012 qualifiers. Zambrotta played his last match in Italy colours in a European qualifier against Serbia in Genoa on 12 October 2010, as his team’s captain. The match was initially delayed, and eventually abandoned after only six minutes due to crowd trouble, with the score at 0–0, after some of the Serbian fans had thrown flares onto the pitch; Italy were later handed a 3–0 victory by UEFA. This match was Zambrotta’s 98th and final Italy cap, making him his nation’s eighth-most capped player of all time. In total, he scored two goals for Italy between 1999 and 2010.

Milan decided not to renew the contracts of several of their veteran players and Zambrotta was one of those along with Filippo Inzaghi, Mark van Bommel, Alessandro Nesta and Gennaro Gattuso. He played his final game for Milan against Novara on 13 May 2012.

After becoming a free agent, in December 2012 Zambrotta began training with his hometown club Como while he was waiting to receive offers from other clubs; Zambrotta had previously been named the club’s honorary president in 2007. In 2013, he began to work on obtaining his UEFA Pro Coaching Licence in Coverciano, Florence.

In 2012, Zambrotta worked as a pundit for the Swiss-Italian broadcasting station RSI. In November 2012, in an interview with Rai Radio 1, Zambrotta revealed that, in the summer, he purchased a family holiday home near Dublin, Republic of Ireland and stated that he would be “open to the idea” of playing in Ireland’s Airtricity League.

On 19 July 2013, Zambrotta signed with Swiss club Chiasso, with an option to become a player-assistant manager during his second season with the club, under Ernestino Ramella. He made his debut with the team on 11 August 2013, in a 0–0 draw against Lugano, in the 2013–14 Swiss Challenge League. On 27 November, following the dismissal of manager Ryszart Komornicki, with the club in last place, Zambrotta was named player-manager; at the end of the season, he helped Chiasso avoid relegation on 14 May 2014, also announcing the end of his playing career. He continued to manage Chiasso the following season, but was eventually sacked on 6 April 2015.

In 2014, Zambrotta opened a fitness complex in his hometown of Como, called the Eracle Sports Centre.

In 2015, Zambrotta took part in and won the RAI 2 reality television game show Monte Bianco – Sfida verticale.

On 14 June 2017, Delhi Dynamos announced that they had mutually terminated their contract with Zambrotta as he prepared to take the role of assistant manager at CSL side Jiangsu Suning.