He played in Major League Baseball (MLB) for 16 seasons and is best known for the 10 years he spent with the Philadelphia Phillies as a right fielder, from 1960 through 1969.

He also led the NL in outfield assists four consecutive times and in double plays once, and ended his career among the top five Phillies in home runs (185) and triples (84).

Manager Gene Mauch was criticized for his handling of the pitching staff over the final two weeks, but players such as slugging rookie third baseman Richie Allen also drew harsh treatment.

He earned two first-place votes for the MVP Award, won by Ken Boyer of the World Series champion Cardinals.

In the 1964 All-Star Game at Shea Stadium in New York on July 7, Callison hit a game-winning walk-off home run off Red Sox pitcher Dick Radatz with two out in the ninth inning, a three-run shot to right field to give the NL a 7–4 victory; it was only the third walk-off home run in All-Star history (and the last one as of 2022), with Callison joining legends Ted Williams and Stan Musial in baseball annals.

But his power production fell off sharply, and he failed to collect 20 homers or 65 RBI in any of his last four Phillies seasons.

In January 1972 he was traded to the New York Yankees, and he found limited playing time over two years, closing his career with a .176 average, one home run, and 10 RBI in 45 games in 1973.