About 30 flights a day pass through the North's airspace to and from the South
Two airlines in South Korea are to re-route flights after North Korea said it could not guarantee their safety.
Pyongyang'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.
South Korea has called on the communist state to immediately withdraw its threat.
"Threatening civilian airliners' normal operations under international aviation regulations is not only against the international rules but is an act against humanity," Unification Ministry spokesman Kim Ho-nyeon said.
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.
The two major airlines, Korean Air and Asiana Airlines, have re-routed some 200 flights over the next two weeks, at the cost of four million won ($2,500; £1,800) per flight.
The annual US-South Korean drill, which involves tens of thousands of troops, starts on Monday and continues for 12 days.
North Korea objects to the exercise every year, but this event comes amid high tensions on the Korean peninsula.
Pyongyang has scrapped a series of peace agreements with the South over Seoul's decision to link bilateral aid to progress on denuclearisation.
Six-nation talks on the aid-for-disarmament deal have stalled and North Korea's neighbours believe it could be preparing to test-fire a long-range missile under the guise of a satellite launch.
Earlier this week, North Korea raised objections to the annual exercise at a rare meeting between its generals and the US-led United Nations Command (UNC) in the South.
Then on Thursday a North Korean committee warned that "security cannot be guaranteed for South Korean civil airplanes" during the forthcoming drill.
No-one knew what "military conflicts will be touched off by the reckless war exercises", it said.
A second short meeting between generals and the UNC was held on Friday, but there were no immediate reports of progress.
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".
America's top envoy on North Korea, Stephen Bosworth, is currently visiting the region in an effort to breathe life into the stalled nuclear disarmament talks.
Speaking in Japan, he described North Korea's warning on South Korean airliners as "unacceptable", and urged the communist nation against any rocket launch, whether of a missile or a satellite.