The incident happened around 8.50pm last night, when a grey Mercedes collided with a black Audi on the A92 at its junction with High Street, Freuchie.
Emergency services attended and the 22-year-old male driver of the Audi was taken to Ninewells Hospital in Dundee with serious injuries.
The 43-year-old male driver of the Mercedes was also taken to Ninewells Hospital for treatment.
Three men, aged 20, 19 and 21, who were all passengers in the Audi were taken to Victoria Hospital in Kirkcaldy for treatment.
The 13-year-old female passenger in the Mercedes was uninjured.
The road was closed for around six hours to allow a full collision investigation to take place.
Constable Kevin Deehan, of Glenrothes Road Policing Unit, said: “This has been a serious crash and our enquiries are ongoing to establish the full circumstances surrounding the incident.
“I would ask anyone who was driving in the area at the time and may have seen either car prior to the crash or anyone with dashcam footage that may assist with our investigation to get in touch with officers.
“Anyone with information can contact police via 101, quoting incident number 3172 of 2 September.”