Save the Children's foreign staff have been ordered to leave Pakistan within two weeks, the aid agency confirms.
It says it has been given no reason for the order, but correspondents say the move is thought be fall-out from the operation that killed Osama Bin Laden.
Following the raid, a Pakistani doctor was arrested for working for the CIA.
Pakistani intelligence officials accuse Save the Children of involvement - the group denies the claims. Six of its staff in Pakistan are foreigners.
The charity has worked in Pakistan for more than 30 years. Correspondents say that it is not thought that the forthcoming expulsions will have any significant impact on its operations in the country in the short term.
Dr Shakil Afridi was arrested after it emerged he had been running a fake vaccination programme on behalf of the CIA as part of efforts to track Bin Laden, who was killed by US special forces in May last year.
The doctor was jailed for 33 years in May in a controversial hearing held behind closed doors under Pakistan's tribal justice system.
It was originally thought that he had been imprisoned for running the fake vaccination programme - but court papers later showed that he was sentenced for alleged links to a banned militant group.
His family have called the treason allegations "rubbish" and his lawyers said they would appeal.
The BBC's Aleem Maqbool in Islamabad says that over the last 18 months foreign staff of other aid agencies in Pakistan have reported increased restrictions on the way they work.
Despite that, huge numbers of Pakistanis have been reliant on their help, particularly following displacement because of conflict in the north-west and after natural disasters, like the floods of the past two years.