McCullough was born in Bellflower, California. She has two older brothers. Her mother is a dance teacher, who took her to rehearsals, and got her involved with acting. McCullough's first appearance was as a 7-month-old baby in a diaper commercial, co-starring with actress Juliet Mills. She started doing gymnastics at the age of 4, and performed as part of a group called the "Gym Dandies." At the age of 6, she auditioned for choreographer Debbie Allen. Allen was eventually won over and cast McCullough in the television series Fame. McCullough followed this up with a dancing part in Breakin' 2: Electric Boogaloo.

In 1985, after failing to land a role on the TV sitcom Webster, McCullough auditioned for the role of Robin Scorpio, the 6-year old daughter of the popular soap couple Robert Scorpio (Tristan Rogers) and Anna Devane (Finola Hughes). The producers made her audition 12 times but eventually gave her the part and her role became a popular success . In 1989 at the age of 11, McCullough won her first Daytime Emmy Award, for "Outstanding Juvenile Female in a Drama Series". One of her most memorable storylines began in 1995, when her teenage character contracted HIV after having unprotected sex with her boyfriend Stone, who was unknowingly infected with the virus and later died of AIDS. She won a second Daytime Emmy in 1996 for this storyline, once again for "Outstanding Younger Leading Actress in a Drama Series".

She took a break from playing the character in 1996 when she went off to college, to study film directing at New York University. In General Hospital, Robin Scorpio was also written out for the same reason, except the character was attending Yale University to study medicine. McCullough continued playing the character during her university breaks.

In 1998, after having briefly returned to the show, she left once again to pursue other acting opportunities, which included co-starring in the primetime drama series Once and Again and Joan of Arcadia. She also wrote and directed the mockumentary Lil Star about girls in childhood beauty pageants, a story which allowed McCullough to draw on her own experiences. After brief returns to General Hospital in 2000 and 2004, she finally returned to General Hospital on a permanent basis in October 2005, with her character Robin Scorpio now a doctor, having graduated from medical school.

In other work, McCullough has danced in other television appearances such as Solid Gold and The People's Choice Awards. She sang backup for the 1989 song "Toy Soldiers" by Martika, and has also had recurring guest-star roles on The Shield. She was also the star of the TV movie Dying to Dance, in which she played opposite Rick Springfield, and Dominic Zamprogna, other General Hospital actors.

All of McCullough's awards and nominations have been for her role as Robin Scorpio on General Hospital.

Tracey E. Bregman, The Young and the Restless (1985) · Ellen Wheeler, Another World (1986) · Martha Byrne, As the World Turns (1987) · Julianne Moore, As the World Turns (1988) · Kimberly McCullough, General Hospital (1989)

Complete list: (1985-1989) · (1990-1999) · (2000-2009)

Cady McClain, All My Children (1990) · Anne Heche, Another World (1991) · Tricia Cast, The Young and the Restless (1992) · Heather Tom, The Young and the Restless (1993) · Melissa Hayden, Guiding Light (1994) · Sarah Michelle Gellar, All My Children (1995) · Kimberly McCullough, General Hospital (1996) · Sarah Brown, General Hospital (1997) · Sarah Brown, General Hospital (1998) · Heather Tom, The Young and the Restless (1999)

Complete list: (1985-1989) · (1990-1999) · (2000-2009)