After three starring junior seasons with the St. Catharines Teepees of the Ontario Hockey Association, Mikita was promoted to the parent Chicago Black Hawks in 1959–60. In his second full year, in 1961, the Black Hawks won their third Stanley Cup. The young centre led the entire league in goals during the playoffs, scoring a total of six.

The following season was his breakout year. Mikita became a star as centre of the famed "Scooter Line", with right wing Ken Wharram and left wingers Ab McDonald and Doug Mohns. Combining skilled defense and a reputation as one of the game's best faceoff men using his innovative curved stick, Mikita led the league in scoring four times in the decade, tying Bobby Hull's year-old single-season scoring mark in 1966–67 with 97 points (a mark broken two years later by former teammate Phil Esposito and currently held by Wayne Gretzky). The 1967–68 season, an 87-point effort from Mikita, was the last year a Chicago player won the scoring title until Patrick Kane's 106-point 2015–16 season.

In his early years, Mikita was among the most penalized players in the league, but he then decided to play a cleaner game and went on to win the Lady Byng Memorial Trophy for particularly sportsmanlike conduct combined with excellence twice. Mikita's drastic change in behavior came after he returned home from a road trip. His wife told him that while their daughter, Meg, was watching the Black Hawks' last road game on television, she turned and said, "Mommy, why does Daddy spend so much time sitting down? The camera had just shown Mikita in the penalty box again.

During his playing career, in 1973, Mikita teamed up with Chicago businessman Irv Tiahnybik to form the American Hearing Impaired Hockey Association (AHIHA), to bring together deaf and hard-of-hearing hockey players from all over the country, and he founded the Stan Mikita School for the Hearing Impaired, inspired by a friend's deaf son who was an aspiring goalie. He also helped bring the Special Olympics to Chicago, bringing his family out to volunteer at races.

Internationally, Mikita played two games of the Summit Series in 1972 for Canada against Soviet Union, both of them in Canada, as well as two exhibition games also during the Summit Series, one against Sweden in Stockholm and one against Czechoslovakia in Prague. He also played several exhibition games for Czechoslovakia in summer 1967 when he came to his country of origin to visit his family.

Awards and accomplishments:

• Ranked 14th all-time in points, 18th in assists, 31st in goals, and 40th in games played (at end of 2017-18 NHL season)
• Won the Hart Memorial Trophy as most valuable player in 1967 and 1968
• Won the Art Ross Trophy as leading scorer in 1964, 1965, 1967, and 1968
• Won the Lady Byng Memorial Trophy in 1967 and 1968
• Stanley Cup champion (1961)
• Named to the NHL's First All-Star Team in 1962, 1963, 1964, 1966, 1967, and 1968
• Named to the NHL's Second All-Star Team in 1965 and 1970
• Played in NHL All-Star Game in 1964, 1967, 1968, 1969, 1971, 1972, 1973, 1974, and 1975
• Won the Lester Patrick Trophy in 1976
• The only player in NHL history to win the Hart, Art Ross and Lady Byng trophies in the same season, doing so in consecutive seasons, in 1966–67 & 1967–68
• Was named to Team Canada for the 1972 Summit Series, but only played two games due to injuries.
• In 1998, he was ranked number 17 on The Hockey News' list of the 100 greatest NHL players.
• Mikita's number 21 was retired by the Blackhawks on October 19, 1980; he was the first player to have his jersey number retired by the Blackhawks.
• Mikita was inducted into the Hockey Hall of Fame in 1983
• Mikita was inducted into the Slovak Hockey Hall of Fame in 2002
• The ice rink in Ružomberok, Slovakia, is named after him.
• In 2011, statues of Mikita and Bobby Hull were installed outside the United Center, where the Blackhawks currently play.
• The first player of Slovak origin who won the Stanley Cup.