The judge ruled that the 2008 settlement that gave $65 million to Cameron and Tyler Winklevoss in cash and stock still stands. The twins had argued that the value of Facebook's stock was more than what Zuckerberg and his team let on.
"At some point, litigation must come to an end," Judge Alex Kozinski, of the US Court of Appeals for the Ninth Circuit in San Francisco, wrote in a unanimous three-judge decision.
"That point has now been reached."
While Kozinski acknowledged the point raised by the twins, he said the plaintiffs
and their lawyers "made a deal that appears quite favourable in light of recent market activity".
In the original suit filed in 2004, the rowing-twins and a third classmate, Divya Narendra, had said that Zuckerberg stole the Facebook idea from them, which was also dramatised in the hit film The Social Network last year.
"The Winklevosses are not the first parties bested by a competitor who then seek to gain through litigation what they were unable to achieve in the marketplace," the judge said.
"And the courts might have obliged, had the Winklevosses not settled their dispute and signed a release of all claims against Facebook," he added.
It is not clear yet whether the twins will take their case to the US Supreme Court.
Facebook is now valued at $50 billion, which is three times more than its value when the settlement was made.