Pakistan has ordered the charity Save the Children to leave the country, with an official accusing the NGO of "anti-Pakistan" activities.
Police have sealed off their offices in Islamabad and foreign staff given 15 days to leave the country.
Save the Children said it "strongly objected" to the action.
Pakistan has previously linked the charity to the fake vaccination programme used by the CIA to track down Osama Bin Laden.
The charity has always denied being involved with the CIA or Pakistani doctor Shakil Afridi, who carried out the programme.
The charity has had no foreign staff in the country for the past 18 months in response to the accusations.
It now has 1,200 Pakistani staff working on projects in health, education and food.
Speaking after the charity was shut, Pakistan Interior Minister Chaudry Nisar Ali Khan said that NGO's were operating beyond their remit with backing from US, Israel and India.
"Local NGOs that use foreign help and foreign funding to implement a foreign agenda in Pakistan should be scared. We will not allow them to work here whatever connections they enjoy, regardless of the outcry," he said in a live television broadcast.
Save the Children, which has operations all over the world, has worked in Pakistan for more than 30 years.
No government explanation
The Pakistani government has not given a formal announcement explaining the decision.
But one official told the AFP news agency: "Their activities were being monitored since a long time. They were doing something which was against Pakistan's interest."
A police official said that the charity's phone calls and offices had been placed under surveillance. Speaking to the Reuters news agency, he added that the charity's activities were "very suspicious".
Condemning the move, Save the Children said it was "raising our serious concerns at the highest levels", adding that its workers were all Pakistani nationals.
A Save the Children official told Reuters that the Pakistan government had been stopping aid shipments entering the country, "blocking aid to millions of children and their families".
It comes after the Pakistani government announced it was tightening the rules for NGOs, revoking several of their licences.
The BBC understands that one of those NGO's, the Norwegian Refugee Council, has ceased all operations in Pakistan as its licence has not yet been renewed.