Two airlines in South Korea are to re-route flights after North Korea said it could no longer guarantee their safety.
The North's threat follows its warnings that a US-South Korean military exercise, due to take place next week, could trigger a military clash.
North Korea has long described such exercises as provocative but tensions between the two Koreas are now high.
About 30 international flights a day usually pass through North Korean airspace to and from the South.
This year North Korea raised objections to the annual exercises at a rare meeting between its generals and the US-led United Nations command in the South.
Tensions are also high in the region amid speculation that the North is planning to test-fire a long-range missile from a base in Hwadae.
On Thursday a North Korean committee warned that "security cannot be guaranteed for South Korean civil airplanes" during the forthcoming military exercises.
It said no-one knew what "military conflicts will be touched off by the reckless war exercises".
The BBC's John Sudworth in Seoul says passenger planes normally leave Seoul for the eastern United States by swinging north over the Sea of Japan to follow the Korean coastline towards Russia and North Alaska.
With flights by Korean Air and Asiana now re-routed, the government has urged North Korea "immediately to withdraw" threats against flights.
"A military threat to the normal operations of civil airplanes not only violates international rules but is also an inhumane act that can never be justified," South Korean's foreign ministry said.
In Washington, US State Department spokesman Gordon Duguid said the North's statement was "distinctly unhelpful".
He said Pyongyang should be working on ways to fulfil its disarmament commitments "rather than making statements that are threatening to peaceful aviation".
Relations between the two Koreas have deteriorated since the election of South Korean President Lee Myung-bak last year.
Mr Lee has ended his predecessors' so-called "sunshine policy" in which the South gave unconditional aid to the North.
Pyongyang has scrapped a series of peace agreements with the South over Seoul's decision to link bilateral aid to progress on denuclearisation.
The annual US-South Korean drill, which involves tens of thousands of troops, starts on Monday and continues for 12 days.
America's top envoy on North Korea, Stephen Bosworth, is currently visiting China, Japan and South Korea in an effort to breathe life into the stalled nuclear disarmament talks.