Thamanat Prompow, secretary-general of the coalition-core Palang Pracharath Party, has been sacked from the position of deputy agriculture minister and Narumon Pinyosinwat, of the same party, has lost her job as deputy labour minister.
The Royal Gazette on Thursday published a royal command removing them both from their portfolios and the cabinet.
The royal command was issued on Wednesday, and stated that the prime minister said it would be appropriate if some ministers were removed for the sake of government.
Prime Minister Prayut responded only briefly to questions, telling reporters he had his own reasons for the changes.
At the parliament, Mr Thamanat told reporters he had signed his letter of resignation from the portfolio on Wednesday because he "cannot share the same path" with the prime minister and national administration did not go the way he expected.
He said he had decided to limit his role to that of an MP for Phayao province for the time being, and would later choose a new political direction and fight.
Mr Thamanat, who was elected PPRP secretary-general only in June, said he had not decided if he would remain with the Palang Pracharath Party.
Mr Thamanat was earlier accused of being behind a move to oust Prime Minister Prayut during last week's censure debate in parliament by organising votes against him.
Political analysts have viewed him as the most powerful political broker in the coalition government. A source at PPRP said Mr Thamanat wanted a ministry, not a deputy's portfolio.
Another source at the PPRP said on Thursday that the prime minister could not tolerate any move against him and an apology from Mr Thamanat was not accepted. Gen Prayut also wanted to closely supervise PPRP himself, the source said.
Gen Prayut had informed Deputy Prime Minister Prawit Wongsuwon, who is the PPRP leader, of the removal of both deputy ministers on Thursday afternoon.
His message reached Gen Prawit after the royal command was published. The source said Gen Prawit had not expected the decision by Gen Prayut and the two men had not discussed the issue earlier.
The source said Mr Thamanat might move to a new party that would be founded in the near future.
Mr Thamanat entered politics with the since disbanded Thai Rak Thai Party in 1999. In 2014 he was a list MP candidate of the Pheu Thai Party.
Ms Narumon is a former spokeswoman for the Prayut government and was appointed deputy labour minister in August last year. She was not available for comment.