Acting speaker Sardar Ayaz Sadiq said 174 lawmakers had voted in favour of the motion, "consequently the vote of no confidence has passed".
No prime mister has ever served a full term in Pakistan, but Khan is the first to lose office through a vote of no confidence.
It was not immediately clear when the assembly will choose a new premier but, opposition leader Shehbaz Sharif was almost certain to be picked to lead the nuclear-armed nation of 220 million people after weeks of high political drama.
Khan, 69, tried everything he could to stay in power -- including dissolving parliament and calling a fresh election -- but the Supreme Court deemed all his actions illegal last week, and ordered the assembly to reconvene and vote.
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