Helgenberger will return as forensic scientist Catherine Willows to help the team solve a decade-old cold case.
According to the network, the episode will involve the investigation into a homicide in the home of a reclusive former casino mogul who was a suspect in a similar crime in 2000.
"We were just really excited to have Marg come back because, obviously, she's such an important part of the history of CSI," executive producer Don McGill said. "And she was super excited. It was kismet!
"The landmark episode will center on a cold case that "takes our characters back to the beginning," McGill explained. "They have to investigate a murder with the same M.O. and the same suspect from 14 years ago."
Helgenberger parted ways with "CSI" last year after 12 seasons on the show.
Her last episode, "Willows in the Wind," aired in January 2012.
"CSI" returns for its 14th season on September 25. The 300th episode will air October 23.