In 2022, Brigitte Macron filed a complaint for libel against two women who posted a YouTube video in December 2021 alleging she had once been a man named "Jean-Michel".
The claim went viral just weeks before the 2022 presidential election.
Brigitte Macron will not attend the hearing on Wednesday and is to be instead represented by her lawyer Jean Ennochi, he told AFP on Tuesday.
Neither he nor Brigitte Macron's team wished to comment before the start of the trial.
Macron, 46, has lashed out at the false information spread about his wife.
He addressed the rumours on International Women's Day, saying, "the worst thing is false information".
"People eventually believe them and disturb you, even in your private life," he said.
The first lady's daughter from her first marriage, Tiphaine Auziere, has said she hopes the trial could quash the "grotesque" rumours.
Messages have multiplied on social media claiming that the 71-year-old first lady, formerly Brigitte Trogneux, is a trans woman whose name at birth was "Jean-Michel."
The two women targeted in the lawsuit, one a self-proclaimed spiritual medium and the other an independent journalist, posted the video along with pictures of the first lady and her family on YouTube in December 2021.
The false claim also led to more serious accusations of child abuse brought against France's first lady.
The disinformation targeting Macron's wife has even spread to the United States where she was attacked in a now deleted YouTube video ahead of the November elections.
Brigitte Macron is among a group of influential women - including former US first lady Michelle Obama and New Zealand ex-premier Jacinda Ardern - who have fallen victim to the growing trend of disinformation about their gender or sexuality to mock or humiliate them.
The president's relationship with his wife 24 years his senior, whom he met while she was a teacher and he was still a teenager, has been a source of media attention in France and abroad.
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