His eldest, Marina, who heads the family's holding company Fininvest, and Pier Silvio, deputy chairman of Berlusconi's media empire Mediaset, both issued angry press releases.
"This country and this democracy should be ashamed of what my father is suffering," said Marina, 47, who has denied persistent rumours that she could succeed Berlusconi.
"My father is no longer a senator but today's vote will certainly not affect his leadership," she said, adding that the expulsion was "a stain that will weigh on our history".
Pier Silvio, 44, said he was "deeply hurt" by the vote.
"I know who my father really is and above all what he has done for Italian businesses and for our country," he said.
The 77-year-old Berlusconi, a three-time former prime minister, left his Rome residence shortly after the vote for his villa near Milan, to be with his family.
Marina and Pier Silvio are both children from Berlusconi's first wife, Carla Dall'Oglio. Barbara Berlusconi, 29, the eldest child from his second marriage to Veronica Lario, also voiced her anger at the Senate ejection.
"With my father's violent expulsion his political rivals think they have an easy road to power," she said.
"This is a political operation that will come back to haunt those who have carried it out," she added.
Berlusconi's children rarely take part in his political activities and manage his vast business holdings – from television and publishing to the AC Milan football club.
There has long been speculation that they could follow their father's footsteps into politics, although Berlusconi himself on Wednesday vowed to fight on.