The participant, who has not been named, was treated by paramedics close to Avonbridge, near Falkirk, but later passed away. Their next of kin have been informed.
A statement from Pedal for Scotland released tonight said: “Our heartfelt condolences go to their family and friends at this time.”
The closed-road cycle marked the event’s 20th anniversary and was the last of its kind.
Organisers said it had more than achieved its aims over the last two decades – encouraging more people to cycle, raising the profile of cycling in Scotland and providing a platform for raising millions of pounds for good causes.
On the Pedal for Scotland Facebook page, Hugh McAninch wrote: “So so sad to learn this that this lovely man lost his life on this day and the devastating impact on his wife and family - our thoughts are with the family at this time.”
Donald Pretsell posted: “So very saddened to read this and my thoughts are with the family. I passed when the emergency services were first in attendance and I, like many, was hoping for a positive outcome.”
Irene McIntosh added: “This is very sad news. Our family had a lovely day today doing the Wee Jaunt and it’s awful to think this was happening. Thinking of the family at this time.”