Nasreen Jalil, a leader of the Muttahida Qaumi Movement, Mr Khan's largest parliamentary ally with seven seats, said the party was joining an opposition bloc seeking to dismiss him in a no-confidence vote.
With the support of the MQM, the opposition bloc potentially has the required number of votes needed in parliament to dislodge Mr Khan and his government, putting his future in doubt.
The former cricket star was expected to address the nation on Wednesday evening, as weeks of political turmoil come to a head.
No prime minister in Pakistan's history has seen out a full term, and Mr Khan is facing the biggest challenge to his rule since being elected in 2018, with opponents accusing him of economic mismanagement and foreign-policy bungling.
Debate on the no-confidence motion is due to start on Thursday, leaving Mr Khan scrambling to keep his own Pakistan Tehreek-e-Insaf (PTI) members on side – as well as a slew of minority parties.
More than a dozen PTI politicians have indicated they will cross over to the opposition, although party leaders are trying to get the courts to prevent them from voting on Sunday.
In the past, Pakistan's political parties have resorted to physically preventing legislators from voting against key legislation by blocking access to the national assembly, leading to cat-and-mouse chases and even accusations of kidnapping.
Senior MQM leader Faisal Subzwari on Wednesday tweeted that his party had finalised an agreement with the opposition, led by the Pakistan People Party (PPP) and Pakistan Muslim League (PML-N).
Hours later, MQM heavyweight Syed Amin-Ul-Haque announced his resignation as technology minister in Mr Khan's Cabinet.
The PML-N and PPP dominated national politics for decades until Mr Khan forged a coalition against the usually feuding dynastic groups.
He was elected after promising to sweep away decades of entrenched corruption and cronyism, but has struggled to maintain support while faced with rising inflation, a weak rupee and spiralling debt.
Some analysts say Mr Khan has also lost the crucial support of the military – claims both sides deny – and Pakistan's army is key to political power.
There have been four military coups – and several unsuccessful ones – since Pakistan gained independence in 1947, and the country has spent more than three decades under army rule.
Pakistan 'heading towards something unpredictable'
If Mr Khan loses next week's vote, a new government could be led by the PML-N's Shehbaz Sharif, the brother of former prime minister Nawaz Sharif, who has not returned since being released from jail to get medical treatment abroad.
Also expected to be given a senior role is the PPP's Bilawal Bhutto Zardari, son of assassinated former prime minister Benazir Bhutto and former president Asif Zardari.
One card up Mr Khan's sleeve could be to call an early election – the next one must be held before October 2023.
"The best option in this situation would have been fresh elections to enable the new government to handle economic, political and external problems faced by the country," said political analyst Talat Masood, a retired general.
"The country is heading towards something unpredictable ... where there is going to be a lot of chaos and problems."
Hassan Askari, another political analyst, agreed.
"The long-term political repercussion of the evolving situation will be instability, continued conflict in politics and inability to cope with economic challenges that Pakistan is currently facing," he said.
Mr Khan has railed against his domestic opponents for weeks, but on Sunday told a rally in the capital that a "foreign conspiracy" was also plotting his removal.
"We have been threatened in writing but we will not compromise on national interests," he said, but offered no evidence or details.