Mackey was drafted from Syracuse University by the Baltimore Colts in the 1963 NFL Draft. He went on to play a total of 10 NFL seasons as tight end, and became known for his size and speed. Mackey played his first nine seasons with the Colts before leaving the team in 1971. He played his final season with the San Diego Chargers, retiring at the end of the 1972 season. Although a knee injury forced him into early retirement, Mackey only missed one game in his whole career.
During his 10 seasons in the NFL, Mackey scored 38 touchdowns and caught 331 passes for 5236 yards.
In his final season as a Colt, Mackey played in Super Bowl V on January 17, 1971. He was involved in a famous game-changing play where he caught a record-setting 75 yard pass from quarterback Johnny Unitas after the ball was deflected twice, once by fellow Colts player Eddie Hinton and once by opposing Dallas Cowboys defenseman Mel Renfro. Baltimore won the game 16–13, following a 32-yard field goal by Jim O'Brien with five seconds left.
During his playing career, Mackey played in five Pro Bowls, including one his rookie season. He was also named All-NFL three times. In 1992, Mackey was inducted into the Pro Football Hall of Fame, becoming only the second pure tight end to be awarded this honor.
Mackey has been included in several lists of great NFL players. In 1999, The Sporting News ranked Mackey at 48 on their list of "The 100 Greatest Football Players." He also placed at number 42 on the NFL Network's list of the "Top 100 Football Players" in 2010.
In his first year as president, Mackey organized a strike following a lockout by owners, with NFL players seeking additional pension contributions and insurance benefits, as well as higher pre- and post-season pay. The strike resulted in increased fringe benefits for NFL players totalling more than $12 million. According to former teammate Ordell Braase, Mackey "had a vision for that job, which was more than just putting in time and keeping the natives calm. You don't get anything unless you really rattle the cage." In 1972, Mackey became the lead plaintiff in a court action which led to the overturning of the so-called "Rozelle Rule," which limited a player's ability to act as a free agent. In 1976, the Rozelle Rule was ruled to violate antitrust laws in Mackey v. NFL.
Several years after retiring from the NFL, Mackey began to suffer from symptoms of dementia. His condition eventually worsened, and his family was forced to put him into a full-time assisted living facility. Although Mackey received a small pension, it was not sufficient to cover the costs of his care, leading his wife Sylvia to reach out to NFL commissioner Paul Tagliabue.
Once made aware of the problem, Tagliabue and NFLPA executive director Gene Upshaw responded with the "88 plan" in February 2007. Named for Mackey's jersey number, the plan provides $88,000 per year for nursing home care and up to $50,000 annually for adult day care for former NFL players, including Mackey, suffering from dementia or Alzheimer's. Mackey died July 6, 2011 at the age of 69.
*Tied for 1st place in their division, but since there was no tie-breaking system in 1965, a game was played to determine who went to the conference championship. The Colts lost the divisional playoff game.