Miami Dade CSI Head Investigator Lieutenant Horatio Caine played by David Caruso, is the Head of the Miami-Dade crime lab, a forensic analyst and former bomb squad officer. In season 1 episode 2, Caine revealed that everything he knew had been taught to him by his mentor, Al Humphreys (played by Lou Beatty, Jr.), who is killed by an explosion while trying to defuse a bomb in the episode. Caine was briefly married to Eric Delko's sister Marisol, which ended when she was murdered by a Mala Noche sniper. He often wears polarized sunglasses.

Miami Dade CSI Level 3 Detective Calleigh Duquesne played by Emily Procter, is a ballistics specialist. Her father is an alcoholic attorney who has tried to rehabilitate himself several times. She has a brief relationship with John Hagen in season 2; in the season 3 finale Hagen shoots himself while she is getting a different gun, after having pulled a gun on her earlier in the episode. In season 4, there were hints at some sort of relationship with Special Agent Peter Elliot. At the beginning of Season 5, she is temporarily in charge of the lab while Horatio and Delko are in Brazil. She appears to be very good friends with fellow CSIs Eric Delko and Ryan Wolfe. Duquesne had a brief relationship with undercover agent Jake Berkeley.

Miami Dade CSI Level 3 Detective Eric Delko played by Adam Rodriguez is a fingerprint and drug identification expert of Cuban and Russian descent. In episode 410, "Shattered", Delko's job is endangered when he is arrested for drug possession, but it turns out he had been buying the drugs for his sister Marisol, to ease the pain of her leukemia treatments. Delko's sister was murdered by a Mala Noche sniper after she married Delko's boss, Horatio Caine. He is also the team's underwater recovery expert. While trying to rescue a woman kidnapped by escapee Clavo Cruz, Delko was critically wounded by one of Cruz's henchmen. He survived, and is now back on the team.

Miami Dade Medical Examiner Dr. Alexx Woods played by Khandi Alexander, is a Miami-Dade medical examiner. Alexx began her medical career in New York as a physician and became a medical examiner with the CSI team after moving to Miami for personal reasons. She often talks to the dead bodies as she examines them, usually as a way of giving them comfort in death. She is married with a young son and daughter. Alexx is not the chief medical examiner, as proved in one the episodes when she was offered to transfer to the night shift by the Chief Medical Examiner, but rejected.

Miami Dade CSI Level 1 Detective Ryan Wolfe (Season 3- played by Jonathan Togo, Ryan was working as a police officer at the time he was hired by Horatio, who noted with approval that Ryan kept his firearm immaculately clean (his predecessor, Speedle, died in the line of duty as a direct result of his poor gun maintenance). He first appeared in the episode "Under the Influence", but did not become a regular cast member until "Hell Night". Ryan appears to believe in curses ("Curse of the Coffin"). Ryan was impaled in the eye with a nailgun in the episode "Nailed". At the end of the episode "Burned" (#522), Ryan was fired for being directly linked to a murder suspect and lying about it. He did appear in the following episode as a crime scene expert and former CSI on TV. In that episode, he apologized to Horatio and will possibly be allowed to return to the team after reviewing all the files from his previous cases (episode #523, "Kill Switch").

Miami-Dade Police Department Homicide Detective Lieutenant Francis 'Frank' Tripp (Season 5- played by Rex Linn, is a Texan homicide detective who regularly accompanies the team to the crime scenes. He has an appreciation for the capabilities of the CSI's work and their excellent results. He has a good working relationship with all the CSIs, particularly Calleigh. He is divorced, with three kids (episode 118, "Dispo Day"). Made regular in season 5, recurring previously.

Miami Dade CSI Trainee Natalia Boa Vista (Season 5- played by Eva LaRue, a DNA analyst who upon first arriving, was only allowed to work on cold or unsolved cases due to the restrictions of her federal grant. She and Delko dated briefly. They broke it off after a pregnancy scare. She escaped an abusive marriage before joining the crew, when her association with a society for battered women was key to helping the team solve the murder of a woman (who had been killed by the victim's ex-husband) assuming an alias for protection. It was revealed at the end of the fourth season that she was the mole in the lab, but the negative information soiling the lab's reputation was not leaked by Boa Vista but by Assistant State Attorney Monica West, and Boa Vista had only reported information to the Feds that showed the lab in a positive light. In season 5, she, much to her distress, discovered her abusive ex-husband, Nick Townsend, was out of prison when he served her with a restraining order; he was working for a private company that cleans up crime scenes, which created a difficult situation with forcing her to make terms with him to get the restraining order dropped. The two shared a tense professional relationship until Nick's murder, a crime for which Boa Vista was briefly considered the prime suspect.