Larijani won 147 out of 279 votes cast during the Wednesday's parliamentary session, while his challenger - the reformer Mohammad Reza Aref - won 123 votes.
In a subsequent vote, Massoud Pezeshkian, a reformist MP, and Ali Motahari, a conservative, were re-elected as the vice-speakers of Iran's parliament.
Larjiani, a moderate conservative, has managed to retain the speakership for over a decade, despite growing support for reformist politics over recent years.
This is in part due to his support for the Iran nuclear deal in 2016, which many hardliners vehemently opposed and subsequently lost their seats during that year's parliamentary elections.
The 58-year-old has publicly slammed US President Donald Trump's withdrawal from the deal, saying he does not have the "mental capacity" for his role as president.
"Trump's abandoning of the nuclear deal was a diplomatic show... Iran has no obligation to honour its commitments under the current situation," Larijani said on 8 May.
"It is obvious that Trump only understands the language of force."
Larijani, who will turn 59 on 3 June, is a member of of a famed Shia clerical dynasty and a regime veteran.
He was a prominent figure in the elite Revolutionary Guards during the 1980-88 war with Saddam Hussein's Iraq and served as state broadcasting chief from 1994 to 2005.
He stood unsuccessfully against hardliner Mahmoud Ahmadinejad for the presidency in 2005 and two years later resigned as secretary of the Supreme National Security Council in protest at his policies which triggered an economically crippling showdown with the West.
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