Glau was classically trained as a ballerina and also studied tango and flamenco. A professional dancer since age 12, Glau relocated to Los Angeles in 2002. She appeared in a few television commercials, but Glau's first television credit was a guest role as a ballerina on the 2002 episode "Waiting in the Wings" (episode 3x13) of the television series Angel. There she caught the eye of producer Joss Whedon, who later cast her in Firefly as River Tam.

Glau appears on the episode "Love Conquers Al" (1x06) of the TV series Cold Case. She has a small role in the film Sleepover, in which she plays a high school senior named Shelly. Summer appeared in the CSI episode "What's Eating Gilbert Grissom?", and the second season of The Unit; portraying Crystal Burns, the girlfriend of Jeremy Erhart (Daniel Wisler).

Glau plays Tess Doerner, a paranoid schizophrenic returnee in the second season premiere of The 4400 (episode "Wake-Up Call"), and the season three episodes "The Ballad of Kevin and Tess", "Terrible Swift Sword" and "Fifty-Fifty". Her character has the power to compel people to do whatever she wants. Glau stars in the science fiction film, Mammoth, which was filmed in Baton Rouge, Louisiana, U.S., and Bucharest, Romania.

Glau was voted Best Actress of 2005 by readers of SFX magazine for her portrayal of River Tam in Serenity. Glau also won a 2006 Saturn Award for Best Supporting Actress in Serenity and was runner up for Best Actress/Movie in the SyFy Genre Awards for 2006.

Glau was cast in a TV movie on ABC Family called The Initiation of Sarah.

In February 2007 it was announced that Glau would be playing the role of Cameron Phillips in The Sarah Connor Chronicles. This character is a Terminator infiltration unit sent back to protect John Connor and Sarah Connor from Skynet.