Pakistan has ordered international charity ActionAid, which supports a range of development and women's rights projects, to shut down its operations in the country, the aid group said on Thursday.
A letter from Pakistan's interior ministry sent to ActionAid, and shared with AFP by the group, said it must cease its work within 60 days.
No representative of the interior ministry was immediately available for comment.
Pakistan first told ActionAid to leave last year after its application to register under new rules for international NGOs was declined, but the group filed an appeal.
That appeal has been denied but ActionAid can apply for registration again in six months, according to the letter, which was delivered to the organisation on Wednesday.
The aid group called the latest notice "a worrying escalation of recent attacks on civil society, academics and journalists".
"We are taking legal advice on potential next steps. In the meantime we are providing support to our staff and partners on the ground," Adriano Campolina, chief executive of ActionAid International, said in a statement.
Seventeen other international aid organisations fear they will face similar action, a senior official of the Pakistan Humanitarian Forum, an alliance of international NGOs, told AFP on condition of anonymity.
In 2015, Pakistan asked all foreign aid and advocacy organisations to re-register with the government in order to enhance the monitoring of their operations.
And last year, a host of international organisations were asked to wrap up operations.
There has been increasing suspicion of foreign aid groups in Pakistan in recent years.
In 2012, a Pakistani intelligence report linked the aid group Save the Children to the CIA's efforts to locate Al Qaeda chief Osama bin Laden.
Save the Children has always denied it had any links with Afridi or the CIA.
But the charity's expat staff were forced to leave Pakistan after the accusations emerged.
Pakistan has since hardened its policies towards international aid groups, accusing them of being covers for spying operations, and has repeatedly warned them to restrict their activities.
