The 91-year-old Fox Corporation owner will appear in a $1.6 billion defamation lawsuit on Thursday and Friday in a Los Angeles courtroom.
Dominion Voting Systems is pressing charges over its claim that Fox amplified claims made by Mr Trump that its machines were used to rig the election in favour of his rival, and eventual winner, Joe Biden.
Accusations of foul play led to Mr Trump supporters storming the Capitol in January 2021 in protest.
"From the highest levels down, Fox knowingly spread lies about Dominion," the election machine company said in a statement.
Fox has argued that it had the right to report the claims of Trump - who the right-wing network frequently covered in more favourable terms than other US stations.
A statement from the company read: “There is nothing more newsworthy than covering the president of the United States and his lawyers making allegations of voter fraud."
Nevertheless, Fox failed an effort to convince a judge to throw out the case in December 2021. Mr Murdoch will be the highest profile person to be questioned now the proceedings begin, after being put back from December 13 and 14 last year.
Dominion has also sought communications from Murdoch, his son Lachlan Murdoch and other Fox News personnel, as it seeks to prove that the network either knew the statements it aired were false or recklessly disregarded their accuracy. That is the standard of “actual malice” that public figures must prove to prevail in defamation cases.
Doug Mirell, a defamation lawyer who has followed the litigation, said he believes Dominion has an "air-tight" case for actual malice because Fox hosts pressed forward with vote-rigging allegations "well after it was quite clear that these claims were demonstrably false."
Dominion alleged in its March 2021 lawsuit that Fox amplified the false theories to boost its ratings and stay abreast of hard-right competitors including One America News Network, which Dominion is also suing.
The complaint cited instances in which Trump allies like Rudy Giuliani and Sidney Powell appeared on Fox News and falsely claimed Dominion software may have manipulated vote counts in favour of Mr Biden.
A five-week trial in the case is scheduled to begin on April 17.