Santo made his debut with the Cubs on June 26, 1960. He played with the team until 1973, then finished his career with the cross-town Chicago White Sox in 1974. During his 14-season run with the Cubs, Santo hit 337 home runs; he was the first third baseman to hit over 300 home runs and win five Gold Gloves, a feat since matched by only Mike Schmidt, a Hall of Fame player with a lifetime batting average 10 points below Santo's .277, in an era noted for weaker pitching.

In the early years of his playing career, he carefully concealed the fact that he had Type 1 diabetes. He feared that had this information come out, he would be forced into retirement. As part of the publicity surrounding "Ron Santo Day" at Wrigley Field on August 28, 1971, he revealed his struggle with diabetes. He was diagnosed with this disease at the age of 18, and was given a life expectancy of 25 years. Santo has had both his legs amputated below the knee as a result of his diabetes; the right in 2001 and the left in 2002. Today, he is a Cubs broadcaster on WGN radio with play-by-play announcer Pat Hughes. He has also worked with Harry Caray, Thom Brennaman, Wayne Larrivee, and Bob Brenly.

Santo has been endorsing the Juvenile Diabetes Research Foundation's annual Ron Santo Walk to Cure Diabetes in Chicago since 1974, and has raised over $50 million for the Juvenile Diabetes Research Foundation (JDRF). In 2002, Santo was named the Juvenile Diabetes Research Foundation's "Person of the Year." Santo also inspired Bill Holden to walk 2,100 miles, from Arizona to Chicago, to raise $250,000 for diabetes research.

On September 28, 2003, Santo's #10 was retired by the Cubs organization, making him the third player so honored behind his teammates Ernie Banks (#14) and Billy Williams (#26). Other prominent Cubs had worn number 10 after Santo's retirement, notably Dave Kingman and Leon Durham. The most recent wearer had been interim manager Bruce Kimm, just the previous year.

In 2005 he came within eight votes of election to the Baseball Hall of Fame by the Veteran's Committee. Bill James, arguably baseball's most respected statistical guru, feels Santo's elevation to the Hall of Fame is long overdue.

Although disappointed at being bypassed, on the day his number was retired, the ever-optimistic "old Cub" told the cheering Wrigley crowd, "This is my Hall of Fame!"

In 2004, Santo and his battle against diabetes was the subject of a documentary, This Old Cub. The film was written, co-produced, and directed by Santo's son, Jeff.