She is well-known for her role as Jeanie Boulet on the medical drama ER (1995–1999, 2008), for which she was twice nominated for an Emmy Award, and for portraying Elizabeth Keckley in the 2012 Steven Spielberg–directed film Lincoln.

Reuben was the subject of a segment of Henry Louis Gates Jr.'s television genealogy series Finding Your Roots.

Reuben came to prominence on the American television series ER as Jeanie Boulet, an HIV-positive physician assistant on the hospital's staff.

[12] Reuben again held a major role in a television series when she starred as FBI agent Brooke Haslett in 1-800-Missing (2003–2004).

[13] Reuben guest-starred in season 12 of NBC's Law & Order: Special Victims Unit, reprising her role as U.S. Attorney Christine Danielson.

In 2012, Reuben played Elizabeth Keckley in the historical drama film Lincoln and her portrayal of the character received critical praise.

The following year, she won the Lucille Lortel Award for Outstanding Lead Actress, the preeminent honour for off-Broadway productions, for her portrayal.

Her latest album, For All We Know, was released on February 14, 2020, on the MCG Jazz recording label, where she is accompanied by guitarist and Grammy winning producer Marty Ashby.

In large part due to her role as an HIV-positive character in ER, she continues to raise awareness about the HIV/AIDS pandemic, lobbying for increased advocacy and speaking at several major fundraising events.

[25] She also takes great interest in global issues, predominantly climate change, nature, and human rights.

[26] Gloria Reuben is the president of Waterkeeper Alliance, an organization that strengthens and grows a global network of grassroots leaders protecting everyone’s right to clean water.