Ackles was born in Dallas, Texas to Roger Alan Ackles and Donna Joan Shaffer. After modeling off and on as a young child, he began to concentrate on an acting career in 1996 upon his graduation from Lloyd V. Berkner High School in Richardson, Texas. In 1997 he received a role on the NBC soap opera Days of Our Lives as Eric Brady, for which he won a 1998 Soap Opera Digest Award for Best Male Newcomer.

After leaving Days in 2000, Ackles went to Vancouver, joining the cast of the James Cameron television series Dark Angel. His first appearance on the show was in 2001 as Max's (played by Jessica Alba) disturbed brother Ben/X5-493. After Ben died, Ackles returned to the series in the second season as Ben's clone, Alec/X5-494, who eventually teamed up with Max and stayed until the show's demise in 2002.

In 2003, he made several guest appearances as C.J. on the hit WB Network show, Dawson's Creek. In 2004, Ackles returned to Vancouver and became a regular on the popular WB Network series, Smallville, which revolves around the life of the teenaged Clark Kent/Superman. His character, Jason Teague, was an assistant coach for the high school football team and a love interest of Clark Kent's ex-girlfriend, Lana Lang, played by Kristin Kreuk.

Ackles returned to television in 2005 in the new WB horror series Supernatural in the role of Dean Winchester, who teams with his brother Sam (Jared Padalecki) to road-trip across the country investigating the paranormal.

Ackles has an older brother, Joshua, and a younger sister, Mackenzie. His interests are baseball, horse riding, and playing guitar.