Five aid groups have been told to leave the North by the end of March, the State Department and aid groups said.
Last year, the UN World Food Programme said that almost nine million people - more than one-third of the North Korean population - was in need of food aid.
The aid block comes amid rising tension over the North's border closures and planned "satellite" launch.
A four-day border closure by North Korea of the shared Kaesong industrial zone left 400 South Korean workers stranded.
And the North has remained defiant throughout joint South Korean-US military exercises, as well as insisting on launching what it says is a satellite early in April.
Aid blocked
Joy Portella, a spokeswoman for one of the groups affected by the decision to stop accepting food aid, Mercy Corps, said they were ordered to leave with any reason being given.
"North Korea has informed the United States that it does not wish to receive additional US food assistance at this time," state department spokesman Robert Wood told reporters in Washington.
"We are obviously disappointed," Mr Wood said. "Clearly this is food assistance that the North Korean people need. That's why we are concerned."
Five US non-governmental organisations distributing food said their 16-member team, which had a mandate to be in North Korea until June, would be leaving by the end of the March.
Under a deal reached in June last year, the United States agreed to distribute 500,000 metric tonnes of food to North Korea - 400,000 through the UN World Food Program and the rest through the NGOs.
Ms Portella said North Korea was still suffering from "rampant malnutrition."
The other four US-based NGOs involved in food distribution are World Vision, Global Resource Services, Samaritan's Purse and Christian Friends of Korea.
The United Nations said on Monday that 6.9 million North Koreans have not received food aid they desperately need.
Hundreds of thousands of people died in the reclusive state in a famine in the 1990s, and the North has relied on outside food aid ever since.
Border woes
Meanwhile, South Korea has expressed its concerns for the future of the Kaesong Industrial Complex, after a four-day border closure by North Korea that left 400 South Korean workers stranded.
"We are at this point not considering shutting down the Kaesong industrial zone," Unification Minister Hyun In-taek told a forum of journalists.
"But if the North repeats the border traffic suspension after the end of the [joint US-South Korean military] drills, the government will consider it a very grave situation and will take appropriate measures," Mr Hyun said.
He said much more was at stake in the Kaesong project than money invested by the 101 companies operating there.
"I believe the Kaesong situation has dashed the hopes that the North and the South would embrace each other despite the grave military and security conditions between the two," Mr Hyun said.
North Korea's announcement that it will launch a communications satellite between 4-8 April has added to the tension on the Korean peninsula.
Analysts believe the satellite launch is actually a planned long-range missile test and the US and its allies have called for it to be cancelled.
Pyongyang recently put its military on full combat alert and shut its border with the South, in what it said was retaliation for the recent annual military exercise by US and South Korean forces.
In January, the North scrapped a series of peace agreements with the South over Seoul's decision to link bilateral aid to progress on de-nuclearisation.
