Pakistan has ordered Save the Children to leave the country, with an official accusing the charity of "anti-Pakistan" activities.
Police sealed off their offices in Islamabad and gave foreign staff 15 days to leave the country.
Save the Children said it received no warning and "strongly objected" to the action.
Pakistan has previously linked the charity to the fake vaccination program used by the U.S. Central Intelligence Agency to track down Osama Bin Laden.
The charity has always denied being involved with the CIA or Pakistani doctor Shakil Afridi. Afridi carried out the vaccination program and told Pakistani interrogators that the CIA had recruited him through a senior official at Save the Children to help in the hunt for Bin Laden.
Pakistani officials said interim permission for the group to operate in the country had run out May 15.
One official told AFP: "Their activities were being monitored since a long time. They were doing something which was against Pakistan's interest."
A Save the Children spokesman said the group is "raising our serious concerns at the highest levels," adding that it no longer has foreign staff in Pakistan.
The group's expat staff was forced to leave the country after a Pakistani intelligence report emerged in 2012 linking it to the CIA. It now has 1,200 Pakistani staff working on projects in health, education, and food, the charity said.
The ouster comes after the Pakistani government announced it was tightening the rules for nongovernmental organizations, revoking several of their licenses.
Pakistan has hardened its policies towards many international aid groups, accusing them of being covers for spying operations. It has repeatedly warned them to restrict their activities, vowing stern action for any "suspicious" activity.
Based on reporting by BBC, AFP, and New York Times