In the summer of 2009, Dos Santos was selected by FC Barcelona B manager Luis Enrique to form part of the second team. Subsequently, Pep Guardiola called him up to the first team during pre-season in England and the tour in the United States. On 15 August 2009, Dos Santos was called up for his first official game with the first team in the Supercopa de España against Athletic Bilbao, although he was eventually ruled out and saw the game as a spectator. He made his debut with Barcelona B on 5 September 2009 against Mallorca B. The match finished in a 1–1 draw. Jonathan made his debut for Barcelona on 28 October 2009, coming on as a substitute for Seydou Keita in the 80th minute in an away Copa del Rey match against Cultural Leonesa, which Barcelona won 2–0. He made his Champions League debut for Barcelona on 24 November 2009 against Internazionale in a 2–0 win at the Camp Nou when he replaced Andrés Iniesta in the 92nd minute. Due to the lengthy 2009–10 season, many Barcelona first-team players were given breaks towards the end of the year. This meant the first team was going to be a few men short, and hence four Barça B players were called up by Guardiola on 29 December 2009. Among these four were Dos Santos, Thiago, Gai Assulin and Víctor Vázquez. His La Liga debut came on 2 January 2010 when he started against Villarreal CF before being substituted by Iniesta in the 60th minute.

Dos Santos made his debut for the Mexico national team when he started in a friendly against Colombia on 30 September 2009. He was substituted for Patricio Araujo in the 72nd minute; Colombia won the game 2–1. This meant that he represented his national team before debuting for his club team.

On 3 March 2010, Dos Santos played 61 minutes against New Zealand. During the match, off a corner from Cuauhtémoc Blanco, the ball rolled towards him and he shot from outside the box but hit the crossbar. Dos Santos was left out of Mexico’s 23-man final squad for the 2010 FIFA World Cup.

On 26 July 2011, Dos Santos started, and scored a goal, in the 2–2 draw against Sport Club Internacional in the 2011 Audi Cup. The match went to a penalty shootout, where he scored as well. Barcelona advanced to the final, where they beat FC Bayern Munich 2–0. He was part of the starting line-up in both matches, though he played at right back in the final. He made his season debut on 29 August in the 5–0 home win against Villarreal, coming in as a substitute for Cesc Fàbregas in the 70th minute.

On 24 November 2011, Dos Santos signed a contract extension which would keep him at Barcelona until 2015. On 6 December, he appeared in the starting squad and played 58 minutes against FC BATE Borisov for the 2011–12 UEFA Champions League group stage match.

In May 2011, Dos Santos was left out of the 2011 Gold Cup final squad. On 24 May 2011, he was called up to play the Copa América. He was subsequently dismissed from the team due to indiscipline.

On 29 February 2012, Dos Santos returned to the Mexico national team for a friendly against Colombia in Miami, Florida, in which they lost 0–2.

Despite rumours of a transfer to other Spanish clubs such as Real Betis and Málaga CF, and Sevilla FC, newly appointed coach Tito Vilanova officially called up Dos Santos to Barcelona’s first team squad, handing him the number 12 shirt. On 6 April 2013, against RCD Mallorca, he came on as a substitute for Andrés Iniesta in the 50th minute and competed against his brother Giovani dos Santos.

On 9 July 2014, Dos Santos signed a five-year deal with Villarreal, and going to play with his brother Giovani. On 21 December, Dos Santos scored his first La Liga goal in a 3–0 victory against Deportivo de La Coruña.

In November 2014, after a two-year absence with the national team, Dos Santos was called up by coach Miguel Herrera for the friendly matches against the Netherlands and Belarus on 12 and 18 November, respectively.

On 27 July 2017, MLS club LA Galaxy announced the signing of Dos Santos as a Designated Player, reuniting him with his brother, Giovani, at the American club. Villarreal sold Jonathan for a fee reportedly around €5–6 million.

In May 2018 he was named in Mexico’s preliminary 28-man squad for the 2018 FIFA World Cup in Russia, and in June, was named in Mexico’s 23-man squad for the 2018 FIFA World Cup.

On 26 March 2019, he scored his first ever senior international goal, opening the scoring in a 4–2 friendly win over Paraguay. In June 2019, he was named in Mexico’s 23-man squad for the 2019 Gold Cup. He scored the lone goal in a 1–0 victory over the United States in the final. He was subsequently listed in the tournament’s Best XI.