The move opens the possibility that Mr Berlusconi, 73, could stand trial in at least three court cases, including one in which he is accused of corruption.
The judges said immunity violated the principle that all citizens were equal.
Mr Berlusconi said he had expected the ruling as the court was dominated by left-wing judges, and would not resign.
"We have a very organised minority of red [left-wing] magistrates who use justice for a political fight," he told reporters outside his residence in Rome.
"We must govern for five years with or without the law."
"The trials that they will hurl at me in Milan are real farces... but we'll carry on," he said, accusing the court, the media and the president of favouring the left.
"I will spend some hours away from taking care of the government and refute them all as liars," he said. "These things invigorate me, they invigorate Italians. Long live Italy, long live Berlusconi!"
The BBC's Duncan Kennedy, in Rome, says the news has stunned Italy, where Mr Berlusconi has widespread public support, although it has dipped in recent times.
The opposition has already called on him to step down, although it remains unclear how Mr Berlusconi's coalition partners will react, our correspondent says.
When Mr Berlusconi came to office he was facing at least three court cases, including one involving the British lawyer David Mills.
In that case Mr Berlusconi was accused of bribing him to give false evidence. Mills, who said he was innocent, was sentenced in February to four years and six months in prison for corruption.
'Distractions'
Mr Berlusconi and his lawyers had argued that he needed the immunity law to carry out his duties as prime minister, our correspondent says.
The appeal to the Constitutional Court was launched by prosecutors including those from the Mills case.
They contended that immunity put Mr Berlusconi above the law and needed to be reversed.
Mr Berlusconi argued that immunity allowed him to govern without being "distracted" by the judiciary.
This is the second time Italy's highest court has thrown out Mr Berlusconi's bid for immunity, after an earlier attempt in 2004 failed.
Of the Constitutional Court's 15 members, five are selected by the president, five by the judiciary, and five by parliament.
They voted 9-6 to in favour of lifting Mr Berlusconi's immunity, the BBC's Duncan Kennedy says from Rome.
The ruling comes at a time when the prime minister's approval ratings have been eroded by a series of sex scandals and his wife has announced that she is divorcing him.
