Emergency services were called to the A911 from Glenrothes to Windygates, near Markinch, shortly before 13:30 on Tuesday.
The 45-year-old woman was a passenger in an Audi A5 Coupe.
Firefighters cut her free from the vehicle and she was airlifted to Ninewells Hospital in Dundee, where she died at about 19:30.
The male driver of the car had minor injuries.
The lorry driver, also a man, was treated by ambulance staff at the scene.
Ch Insp Davie McCulloch, head of road policing at Fife Constabulary, said it was a "very serious accident" and that the road would probably remain closed until Wednesday afternoon while investigations were carried out.
"It is essential that we get as much relevant information as possible and I am appealing to anyone who saw either the car or the lorry prior to the collision, or the collision itself, to contact us," he added.
