Head of the opposition Democratic Party's faction in parliament, Dario Franceschini, submitted the no-confidence motion on Friday. A date for the vote has not yet been set.
While it appears that that the 74-year-old Berlusconi can no longer win a majority in parliament, he has vowed not to step down.
Earlier in the week, his government lost a vote over a bill on immigration policy after one of his allies, the Future and Freedom for Italy (FLI) party led by Gianfranco Fini, voted against the government.
At an FLI meeting last Sunday, Fini had called for Berlusconi's resignation after the Italian leader once again become entangled in sex scandal allegations, Press TV reports.
"Berlusconi's days are numbered," said Italy of Values politician Leoluca Orlando. "Soon Italy will free itself from a cancer that has destroyed its national economy, covered it with filth and thrown its moral value into the dirt."
Berlusconi's ally-turned-rival and speaker of parliament Gianfranco Fini, who recently created his own Future and Freedom for Italy party, called on the prime minister to resign at a party conference on Sunday, according to Deutsche Welle.