The 63-year-old woman was cycling on Bridge Street, St Andrews, Fife, at about 3.50pm yesterday when she collided with a man on foot.
Both fell on to the road and the cyclist suffered serious head injuries, police said. She was taken to Ninewells Hospital but later died.
The pedestrian was not seriously injured but left the scene before police arrived and officers are trying to trace him.
Sergeant Iain Hutchison said: "Police are still trying to establish the full circumstances surrounding this tragic incident and we are eager to hear from members of the public who were in the area at the time of the collision.
"In particular we would like to speak with the male pedestrian involved in the collision. We would like to stress this is just to assist with our inquiry.
"We are also keen to trace an elderly couple who were walking down Bridge Street at the time of the accident as they may hold vital information as to what happened.
"In addition, anyone with any further information relevant to this investigation is asked to contact police immediately."