The court issued its suspension just as Pita was sitting in parliament for another day of deliberations on whether he could become prime minister, after his first attempt fell 51 votes short last week.
Pita's Move Forward Party (MFP) has ridden high on the hopes of young and urban Thais wearied by nearly a decade of army-backed rule, but its efforts to form a government have stumbled since the May polls.
Thailand's conservative establishment vehemently opposes the party's economic reform platform and its pledge to soften the kingdom's strict royal defamation laws.
On Wednesday, the country's Constitutional Court announced it would take up a case on whether Pita should be disqualified from parliament altogether for owning shares in a media company, ordering him to leave the assembly in the meantime.
"It was commanded that the respondent must suspend his role... until the Constitutional Court has made its decision," the court said in a statement.
Lawmakers are forbidden from owning shares in media companies under Thailand's constitution, though the television station in question has not broadcast since 2007.
Pita, Harvard-educated and wealthy from a family-run agrifood business, has said the shares were inherited from his father.
Under Thai law, Pita remains eligible to stand as a candidate for prime minister but must leave the lower house and will be unable to vote.
Dozens of his supporters, wearing the MFP's tangerine colors, shouted angrily at a heavy police cordon outside parliament after news of the suspension broke.
Few expect his party to have made up last week's shortfall, and lawmakers aligned with the military immediately forced a debate on whether the law allowed Pita to be considered a second time.
Thailand's senate is stacked with military appointees, with only 13 of 249 serving senators voting for Pita last week.
Those who did not support him last week were unlikely to be "brave and courageous enough" to change their minds on Wednesday, Napisa Waitoolkiat, a political analyst with Naresuan University, told AFP.
Pita remained in the chamber immediately after the suspension was issued. He has vowed to step aside to make way for a coalition partner to form a government if his second attempt fails.
'Personal benefits'
Other roadblocks have been thrown in front of Pita's candidacy.
The court has also agreed to hear a case alleging that MFP's campaign promise to amend Thailand's royal defamation law is tantamount to a plan to "overthrow" the constitutional monarchy.
Pita's party has ignored strident opposition to its pledge to revise the law, which can allow convicted critics of the monarchy to be jailed for up to 15 years.
The MFP's reformist platform also poses a threat to family-owned business monopolies that play an outsized role in the kingdom's economy.
Pita conceded Wednesday that lawmakers who gained "personal benefits" from the present order, including those with stakes in powerful Thai enterprises, would refuse to vote for his party.
The Constitutional Court has intervened in Thai politics before.
The billionaire leader of MFP's predecessor party, Thanathorn Juangroongruangkit, was disqualified as an MP in 2019 after falling afoul of the same shareholding rule.
'Justification for crackdown'
If Pita is unable to become premier, the coalition backing him is expected to fall in line behind property tycoon Srettha Thavisin, potentially relegating MFP to serve in opposition.
Srettha's Pheu Thai party is seen as a vehicle for the Shinawatra political clan, whose members include two former prime ministers ousted by military coups in 2006 and 2014.
But as a successful entrepreneur liked by fellow business leaders, the 60-year-old is seen as a potential compromise acceptable to the Thai elite.
Prawit Wongsuwan, 77, a former Thai army chief who served as number two in the junta that took power in 2014, has also been floated as a candidate by parliament's military bloc.
Thai voters roundly rejected army-backed parties in May's election, and political analyst Thitinan Pongsudhirak said the prospect of a military presence in the next government could spark a backlash in a country that is no stranger to political unrest.
"If Move Forward is excluded, there will likely be protests... if protesters overreact, a Pheu Thai-led government would have some justification for a crackdown."