The rejection of the third nominee for the post came after Rouhani, a reputed moderate who took office in August, cautioned against efforts to undermine his government.
"The parliament does its duty within the legal framework, however the method is sometimes in a way that might be seen by ill-wishers as weakening or even undermining the government," Rouhani said, quoted by the official IRNA news agency.
The nominee, Nasrollah Sajadi, received 124 of the 253 votes cast, with 107 against and 22 abstentions, according to state media. He needed at least half of the votes plus one to be approved.
After the rejection, Rouhani picked Mahmoud Goudarzi as his latest nominee for the post.
Mohammad Dehqan, a member of parliament's presiding board, said Rouhani's remarks had "annoyed" the lawmakers, contributing to the snub, the Mehr news agency reported.
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