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The Southeast Asian nation has been in turmoil since an alliance that swept to power in 2018, which was headed by Mahathir Mohamad and included Anwar, collapsed in February amid bitter infighting.
Muhyiddin Yassin became premier at the head of a coalition backed by a scandal-plagued party which had been ousted at the polls two years earlier, but he had only a wafer-thin majority in parliament.
Speaking at a Press conference in Kuala Lumpur, veteran politician Anwar - who has long sought to become prime minister - said he now had the backing of enough MPs to form the government and oust Muhyiddin.
"Conclusively we have a strong, formidable majority," the 73-year-old said. "The government under the leadership of Muhyiddin Yassin has fallen."
But he did not reveal how many MPs were backing him or which parties they were from. A government must command the support of a majority of the 222 MPS in parliament.
The king has the authority to appoint a prime minister in Malaysia, if a candidate can prove he has enough support in parliament.
Anwar said had been granted an audience with the king on Tuesday - but it had been postponed to an unspecified date as the monarch is undergoing medical treatment.
Muhyiddin appeared unshaken by the opposition leader's challenge, and spent most of a nationally televised address outlining new stimulus measures for the coronavirus-hit economy.
At the end of his speech, however, he called on Malaysians to back his government as the country faces "an economic and health crisis.
"I appeal to you to reject the blind actions of some politicians who deliberately want to affect political stability and the national economic plan, which is being implemented well at the moment."
Other members of the government brushed off the move.
International Trade and Industry Minister Azmin Ali brandished Anwar an "incorrigible liar and political psychopath", while Science and Technology Minister Khairy Jamaluddin joked "nothing collapsed or fell".
Anwar's move came ahead of weekend elections for the legislature in the eastern state of Sabah, which will be a major test of the current government's popularity.
Muhyiddin's administration has had the difficult task of leading Malaysia through the coronavirus pandemic, and the economy suffered its worst contraction in more than 20 years in the second quarter amid a strict lockdown.
But restrictions have been eased and authorities have won praise for their handling of the outbreak, which has been relatively small - Muhyiddin received a 69 per cent approval rating in survey earlier this month.
Long-time opposition leader Anwar was a key figure in the alliance that won a shock victory at landmark elections in 2018, toppling a scandal-plagued coalition that had ruled Malaysia uninterrupted for over six decades.
Voters kicked out the old regime in large part due to anger at former premier Najib Razak's involvement in a massive financial scandal which saw billions looted from state coffers.
Mahathir, now 95, became prime minister for a second time and Anwar was released from jail, where he had been serving a sentence after being convicted of dubious sodomy charges.
But the government collapsed amid tensions between rival factions over whether Mahathir would stick to a promise to hand power to one-time nemesis Anwar, and Muhyiddin seized power without an election.
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