He supported Manchester United and his talent was swiftly recognised when he played for England Schoolboys at the age of 15. He achieved a childhood ambition when, in the same year, the club he supported gave him an apprenticeship in September 1959.

He was an unlikely-looking footballer in many ways – he was short, at a time when teenagers were being rejected by clubs purely because of their lack of height; he was also a wearer of dentures, the removal of which prior to matches gave him a gap-toothed expression which had the potential to scare. He became significantly bald at a young age and sported a dramatic comb-over. He also suffered from severe shortsightedness, meaning he needed strong contact lenses when playing and wore thick spectacles off the field.

Nevertheless, Manchester United manager Matt Busby saw something in the tenacious youngster. Stiles was given his debut as a full back in October 1960 against Bolton Wanderers. Stiles' simple passing game and fearless ball-winning skills saw his swift conversion into a "holding" midfield player of a type now a feature of all top teams but still a rarity at a time when forward lines consisted of five players and the midfield was restricted to covering half-backs. Stiles was deployed in the middle of the park to snuff out the flair and peril of creative opponents, and this he did with much success. His ability to gain and retain possession also allowed his more skilled teammates like Bobby Charlton and, later, George Best to utilise more space on the field. Stiles' defensive skills were also utilised by United managers to great effect throughout his successful career.

That said, Busby was not afraid to sacrifice Stiles when required and when United beat Leicester City 3–1 in the 1963 FA Cup final, Stiles was not picked for the game. He began to make more frequent appearance in the seasons ahead, however, and won the First Division title in 1965.

In 1968, he won his second League championship medal with Manchester United, but greater club honours were to come. United reached the European Cup final, which was to be staged at Wembley, and Stiles' old nemesis Eusébio was again someone he had to deal with as part of a fearsome attacking potential posed by opponents Benfica. Stiles' played well without keeping Eusébio wholly quiet, and with the score at 1–1 and just a few minutes left, the Portuguese legend got away from the United defence with just goalkeeper Alex Stepney to beat, yet only managed to slam the ball straight into Stepney's midriff. United won 4–1 and became the first English club to lift Europe's main trophy.

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After 395 matches and 19 goals, Manchester United sold Stiles in 1971 to Middlesbrough for £20,000. Two years later, he became a player-coach at Preston North End when Bobby Charlton was manager.