The collision between Settle and Long Preston, near The Courtyard, is believed to have involved a motorcyclist.
The Yorkshire Air Ambulance landed at the scene.
The road was closed for several hours on Saturday afternoon and re-opened in the evening.
North Yorkshire Police said: "The A65 is closed between Settle and Long Preston while we deal with a serious collision. Please avoid the area, and find an alternative route for your journey, while the emergency services work at the scene."
The force has been contacted for further information.