The remaining members of the cult comedy troupe said they had "reluctantly decided" that their July 20 gig at the O2 in London will "probably be their last live performance together".
A statement released by their publicist said: "The Pythons had hoped to take up some of the many offers to perform at arenas all over the world, but individual commitments to other projects have made this impossible."
The gigs come more than 30 years after their last stage performance.
The Pythons - John Cleese, Michael Palin, Eric Idle, Terry Gilliam and Terry Jones - announced their comeback in November.
Tickets for the final show - one of 10 planned for the London venue - go on sale at noon today.