The original starring cast consisted of Anthony Edwards as Dr. Mark Greene, George Clooney as Dr. Douglas "Doug" Ross, Sherry Stringfield as Dr. Susan Lewis, Noah Wyle as medical student (later Dr.) John Carter, and Eriq La Salle as Dr. Peter Benton. Julianna Margulies guest starred in the pilot as Nurse Carol Hathaway before becoming part of the regular cast. Cast members were regularly added and departed the show starting in the second season, and continuing right up until the end of the series, though most of the original cast remained intact for a number of seasons with some additions.

In addition to the main cast, ER featured a large number of recurring supporting cast not billed as starring, but frequently playing notable roles in many episodes during their tenures. The most common of these roles were those of desk clerks, nurses, and occasionally doctors not part of the main cast. In addition, ER featured a significant roster of guest stars, most frequently portraying the many patients required for each episode. Many notable celebrities guest starred on the show.

Most episodes center on the ER, with almost all scenes at the hospital, but usually include at least one scene outside of the hospital. In addition, most seasons included at least one storyline located completely outside of the ER, often outside of Chicago. One early such instance involved a road trip near Las Vegas, Nevada (Drs. Ross and Greene). Season Eight included a storyline in Hawaii (Drs. Greene and Corday); seasons Nine and Ten included storylines in the Democratic Republic of Congo (Drs. Kovac and Carter); and Season Twelve included a storyline set in the Darfur region of Sudan (Drs. Pratt and Carter, with Noah Wyle appearing as a guest star in the season following his departure from the show).

ER won 23 Emmy Awards, including Outstanding Drama Series (1996), and received 124 Emmy nominations, the most of any television show in history.