The collision, involving a Ford Focus and a Hyundai i30, happened on the A92 between Ladybank and Freuchie shortly before 6am today.
The two occupants of the Ford died at the scene. The man driving the other car was taken to Ninewells Hospital in Dundee for treatment to serious injuries.
The road was closed following the crash to allow police to carry out investigations.
The Scottish Fire and Rescue Service said they were called to the scene by the ambulance service at 5.49am.
Crews from Glenrothes, Lochgelly, Cupar, Auchtermuchty and Dunfermline were dispatched to the scene, where they found two cars with three men trapped.
A service spokeswoman said: "Firefighters immediately got to work to stabilise one of the vehicles and used hydraulic cutting gear to remove the roof and safely free the trapped male casualty.
"The casualty was passed into the care of the ambulance service and taken to Ninewells Hospital with serious injuries.
"Sadly, the two males in the other vehicle passed away at the scene and our thoughts are with their families and friends."
Police have urged anyone with information which could help the investigation to come forward.
A Police Scotland spokesman said: "Police in Fife responded to the A92, near Ladybank, at around 5.50am on Wednesday January 15 following a serious road collision.
"A Ford Focus was involved in a collision with a Hyundai i30 resulting in the two male occupants of the Focus sustaining fatal injuries. They were pronounced dead at the scene.
"The male driver of the Hyundai has been taken to Ninewells Hospital for treatment after sustaining serious injuries.
"Enquiries into the full circumstances surrounding this incident are ongoing and anyone who can assist with our investigation is asked to contact police immediately."