"This is a political trial and if anyone thinks they have weakened me, they are mistaken", Salvini said ahead of the vote.
"It will be a joy for me to go to (court in) Sicily to defend my right to defend our attractive country", Salvini, 47, told the Senate before the vote, knowing that it would go against him.
A prosecutor eventually ordered the seizure and evacuation of the ship, operated by the Spanish group Open Arms.
The anti-immigration, right-wing politician is already set to stand trial in a similar case.
The move clears the way for possible charges against the former interior minister for refusing to allow 164 migrants to get off a ship in Sicily last August.More news: Crude oil falls as USA stimulus talks trough
Under current law, ministers can not be tried for actions they take while in office, unless parliamentary immunity is revoked by a vote in the Senate.
But in another case that went to the Senate while he still served as Italy's minister, Salvini's party voted not to lift his immunity, protecting him from prosecution for not allowing 190 migrants to disembark from another coast guard ship in August 2018.
In February, the Senate voted to strip him of his parliamentary immunity in a case involving the Italian coastguard. "I am proud of it, I would do it again, and I will do it again", Salvini said on Wednesday.
He insisted the decision to stop migrants from getting off the ship - until a deal was reached with European Union countries to take them in - was reached collectively within the government.
The opening court hearing because case has actually been delayed up until 3 October since of coronavirus limitations.More news: Rio Tinto sees profits slide, but eyes US$2.5bln dividend
A recent Demopolis poll found that Salvini's Lega has dropped more than 11 percentage points in a year, from holding 37 per cent of voting intentions to 25.4 per cent today.
"At the moment Salvini generates little media interest". "Those who vote to send him to trial in a bid to create political problems for him could end up giving him the limelight instead".
Mr Salvini is now in opposition but is determined to become prime minister.
Salvini's resistance League celebration remains the most popular in Italy, but it's been slipping in opinion polls.More news: Why Osimhen signed for Napoli despite ‘racism fear’