The pair acquired The Crucifixion for €220,000 in 2008, it having previously sold at auction for £39,000 in 2007.
It is thought to have been produced by a follower of Pieter Brueghel the Younger in around 1618, and is based on an original by the Dutch artist, who died in 1636.
Following a year gathering dust, the brothers began to make changes to the piece, including the transformation of the crowd into a range of devilish monsters.
One man has the number 666 on his back - a reference to Brueghel's nickname "Hell Brueghel", due to his several depictions of the underworld.
It has now sold for £750,000.
The piece was snapped up at the Chapman brothers' exhibition of their work at London's White Cube gallery, which runs until September.
The majority of the exhibition's pieces were produced by the two brothers independently of each other.
Jake told Euronews: "The whole point was not about the show. It was about producing the work."
Dinos continued: "But also actually in the final instance, the idea of it being Jake or Dinos Chapman was of no consequence, because it was so resolutely like the work we make anyway."
The Chapmans remain best known for their depiction of 10,000 miniature Nazis in 2000, although this recent sale suggests their talents are still admired by art lovers.
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