Washington agreed to give its blessing for the funds' release to win progress on the nuclear issue. But North Korea has refused to move forward on a February pledge to start dismantling its nuclear programme until it receives the money.
Although Pyongyang was allowed several months ago to begin withdrawing the cash, the funds remained in the bank in this Chinese territory for reasons that were never made clear.
It was widely believed that other banks didn't want to receive the tainted money.
Pyongyang was also reportedly demanding that the money be transferred via a US bank to prove the funds were clean.
The long-awaited transfer finally happened on Thursday, said Francis Tam, Macau's secretary of economy and finance.
"Banco Delta Asia transferred more than $20 million out of the bank this afternoon in accordance with the client's instruction," Tam told reporters on the sidelines of a business gathering.
But Tam would not say where the money was sent. "We have heard reports in foreign media that the money can be wired via the US or Russia, for example. I think these routings are possible," Tam added.
North Korea had $25 million in the bank, but Tam would not provide a specific figure for how much was transferred.
But he said, "most of the money in this account has already transferred out. There will probably not be another transfer."
If some of the money remains in the bank, it could become a new reason for North Korea to delay progress with the pledge to shut down its nuclear weapons programme.
David Zweig, a political scientist at Hong Kong University of Science and Technology, said that it would be inconsistent of the US to re-circulate funds it described as tainted.
Zweig said that the US treasury department wanted the money to remain frozen, while the US state department wanted it released to make progress on the nuclear issue.
"Clearly this is a ridiculous situation because on the one hand, the US government wants the money to go to North Korea, and the other arm has made it almost impossible to do that. To me, that's all very strange."