"The conditions for continuing our cooperation with this government no longer exist," Paolo Romani, chief senator for Berlusconi's Go Italy (Forza Italia) party, said in a message on Twitter.
The move will not bring down the government as a group of former Berlusconi loyalists earlier this month broke away from the scandal-tainted ex-premier to form their own party.
The New Centre-Right group led by Deputy Prime Minister Angelino Alfano has said it will stay in the coalition with Prime Minister Enrico Letta's centre-left Democratic Party even if Berlusconi is expelled.
Letta's government will now have a much slimmer majority in the Senate of around 10 senators, while it will continue to enjoy strong support in the lower house because of many centre-left lawmakers.
Alfano's party accounts for about 50 parliamentarians.