He is best known for his portrayal of Dean Winchester in The WB/CW dark fantasy drama series Supernatural (2005–2020) and appearing in television series such as NBC's Days of Our Lives as Eric Brady (earning him several Daytime Emmy Award nominations), super soldiers Ben/X5-493 and Alec/X5-494 in Fox's Dark Angel, Jason Teague in The WB/CW's Smallville and Ben/Soldier Boy in Amazon Prime Video's The Boys.

He also starred as the lead in the box office success My Bloody Valentine 3D, and provided the voices of Jason Todd in the animated film Batman: Under the Red Hood and Bruce Wayne/Batman in the animated adaptation of Batman: The Long Halloween, Legion Of Super-Heroes, and Justice League: Warworld.

He appeared in several guest roles on Mr. Rhodes, Sweet Valley High and Cybill before joining the cast of the NBC soap opera Days of Our Lives as Eric Brady in 1997.

He joined the cast of The WB's hit show Dawson's Creek during its final season, playing CJ, Jen Lindley's lover.

Dean and his brother Sam (Jared Padalecki) drive throughout the United States hunting paranormal predators, fighting demons and angels, and showcasing fantasy & sci-fi pop culture.

The show ended after fifteen seasons, making it the longest-running North American sci-fi series in history.

He also appeared on stage from June 5–10, 2007 with Lou Diamond Phillips in A Few Good Men at Casa Mañana Theatre in Fort Worth, Texas as Lt. Daniel Kaffee.

[16] On October 22, 2010, Disney Interactive Studios announced he would be voicing a character named Gibson in the video game Tron: Evolution which was released on December 7, 2010.

[17] After years of performing at conventions, Ackles released his first original song as a professional singer titled "Sounds of Someday" which is the first track from his debut album.

[20][21] He later portrayed a version of Soldier Boy in the "Jumanji" episode of the show's spin-off Gen V who is an imaginary friend of Cate Dunlap.

In May 2022, he guest starred on the ABC drama Big Sky's second season finale "Catch a Few Fish", as temporary sheriff Beau Arlen.

[32] Ackles is a co-owner of a brewery in Dripping Springs, Texas, Family Business Beer Company, with his wife and in-laws.