After modeling on and off since the age of four, Ackles began to concentrate on an acting career in 1996. 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 won a 1998 Soap Opera Digest Award for Best Male Newcomer and went on to be nominated three times (in 1998, 1999, and 2000) for a Daytime Emmy Award for Outstanding Younger Actor in a Drama Series for his work on Days of our Lives.

Ackles departed Days of our Lives in 2000 and went on to appear in the mini-series Blonde, about the life of Marilyn Monroe. He also auditioned for the role of the young Clark Kent on Smallville, a role offered to actor Tom Welling instead. Next he appeared in a guest role on the James Cameron television series Dark Angel on Fox in 2001 as serial killer Ben/X5-493, the brother of main character Max/X5-452 (played by Jessica Alba). His character died in the episode, but Ackles returned to the show as a series regular in the second season as Ben’s sane clone, Alec/X5-494. He remained with the show until its cancellation in 2002.

Ackles worked steadily throughout 2003. He joined the cast of the WB’s hit show Dawson’s Creek during its final season, playing CJ, Jen Lindley’s lover. Afterward, Ackles filmed several episodes of the unaired series Still Life for Fox before it was abruptly dropped. He also had a small role in the 2004 short film The Plight of Clownana. Ackles was the producer’s first choice to play Eliza Dushku’s love interest on the second season of Tru Calling; Ackles turned down the role, which was then offered to Eric Christian Olsen and the character’s name was changed to “Jensen” because the producers of Tru Calling liked Ackles’ name. Ackles returned to Vancouver (where Dark Angel was filmed) in 2004 to become a regular on Smallville playing the assistant football coach Jason Teague, who was also the newest romantic interest for Lana Lang (played by Kristin Kreuk). He also had a lead role in the 2005 film Devour in which Ackles’ father, actor Alan Ackles, also had a role playing the father of Ackles’ character, Jake Gray.

In 2005, Ackles joined the cast of the WB show horror/action series Supernatural where he stars as Dean Winchester. 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 is currently in its fifteenth and final season on the CW, the longest-running North American sci-fi series in history.

In the summer of 2007, Ackles took on the role of Priestly in the independent comedy Ten Inch Hero. The film began hitting the film festival circuit in early 2007 and Ackles received high praise for his comedic timing in the role. In February 2009, the film was released on DVD exclusively by Blockbuster Home Video. 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. Ackles received strong praise for his work in this role, which was also his professional theatre debut.

In the summer of 2008, Ackles was cast in the remake of the cult film My Bloody Valentine 3D, which opened nationwide on January 16, 2009.

After three years of dating, Ackles became engaged to actress and model Danneel Harris in November 2009. The couple married on May 15, 2010 in Dallas. Their first child, a daughter, was born in May 2013. In August 2016, the couple announced that they were expecting twins, a boy and a girl who were born in December.

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.

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 . In 2018, Ackles collaborated with musician Steve Carlson to form Radio Company. Together Ackles and Carlson completed a debut album entitled Radio Company Vol. 1. The album’s first single was released on iTunes and the album was released in November 2019.

In 2019, Ackles was chosen to serve as King Bacchus LI in the Krewe of Bacchus parade.

In August 2020, it was announced he would join the cast of The Boys as Soldier Boy. More recently, he and his wife formed Chaos Machine Productions with a deal at Warner Bros. Television.