Best returned home to join Belfast Harlequins in 2002 where he captained the team and gained his first Ulster contract in 2004. He made his debut for Ulster as a replacement at home to Munster in the 2005 Celtic League. He replaced his brother (now retired Ireland and Ulster prop Simon) as Ulster captain for the 2007/08 season and continues to hold the position. He is renowned for his abilities as a scrummager. Best missed the entire 2009/10 season due to surgery on a chronic disc problem. Best scored two tries in 2011/12 and 2010/11 for Ulster. He was part of the Ulster side that was defeated by Leinster in The Heineken Cup final in 2012.

Best made his international debut for Ireland as a replacement against the All Blacks at Lansdowne Road in November 2005 and made his first start against the Springboks in 2006. He has been involved with the national team ever since, initially sharing the number 2 shirt with Jerry Flannery. He was a member of the Irish teams that won the 2007 Triple Crown and was in the Irish squad for the 2007 Rugby World Cup as well as the 2009 Six Nations Championship which completed the Grand Slam.

He captained the Ireland team that toured North America in 2009, while many of his Ireland teammates were on the British and Irish Lions team that began their tour of South Africa at the same time. He became the first-choice hooker at the 2011 Rugby World Cup after an injury to Jerry Flannery. Best also played his 50th test for Ireland during the World Cup, scoring a try as Ireland beat the United States during the pool stages.

Best was added to the British and Irish Lions squad for the 2013 British and Irish Lions tour to Australia on 26 May after English hooker Dylan Hartley was removed from the squad after receiving a red card in the Aviva Premiership final for abuse of an official on 25 May 2013. In 2014 Best won his 2nd Six Nations title.

In January 2016, Best was named as the new Ireland captain for the 2016 Six Nations Championship. On November 26, 2016, Best became Ireland’s fifth centurion, gaining his 100th cap in a test match vs Australia in the Aviva Stadium, Dublin. The previous week, Best captained Ireland to a historic 40-29 win over New Zealand in his 99th appearance, Ireland’s first ever win over New Zealand.

Best played his 200th game for Ulster in the Pro14 in 2017, earning the honour against Leinster.

After finishing the 2017 Six Nations, Best was selected for the British and Irish Lions for the second time, on the 2017 tour to New Zealand. Best started in four of the five matches he played on tour, captaining the Lions to a 34-6 win over the Chiefs and a 31-31 draw against the Hurricanes. Best was not subbed off in either of those matches, and received an OBE while he was on tour with the Lions.

Best was appointed Officer of the Order of the British Empire (OBE) in the 2017 Birthday Honours for services to rugby.

In 2018 while Ireland Captain, Best received criticism for attending a trial in a sign of support for his teammates on trial for the alleged gang rape of a teenage girl at a party in Belfast, which was ultimately dismissed. He admitted regret in attending the trial a year later in interviews with the press.

On 18 April 2019, Best announced that he would retire from professional rugby after the 2019 Rugby World Cup. “It is with mixed feelings that I announce my retirement from Ulster Rugby as of the end of this season,” said Best. “This feels like the right time for me to go out on my terms, a luxury for which I feel very privileged,”.

His final test for Ireland was on 19 October 2019, when Ireland lost to New Zealand, 14-46 in the quarter-finals of the 2019 Rugby World Cup, which was also Best’s fourth World Cup tournament. Best was replaced by Niall Scannell in the 63rd minute of the test, receiving a deserved standing ovation from the crowd as he departed the field. His achievements across his career earned his subsequent praise from All Blacks Captain, Kieran Read.

Best played his last game professional rugby match with the Barbarians, on 16 November 2019, captaining the team with the Flying Fijians claiming the Killic Cup 33-31.