The pro-EU lawmakers join the so-called Independent Group founded by former Labour members of parliament on Tuesday.
In what is likely to be seen as a rebuke to Prime Minister Theresa May's efforts to unite the party, Anna Soubry, Sarah Wollaston and Heidi Allen announced their resignation in a joint statement on Wednesday.
"We no longer feel we can remain in the party of a government whose policies and priorities are so firmly in the grip of the ERG and DUP," they said, referring to a group of pro-Brexit lawmakers in the Conservative Party and the Northern Irish Democratic Unionist Party which props up the government in parliament.
"Brexit has re-defined the Conservative party — undoing all the efforts to modernize it. There has been a dismal failure to stand up to the hardline ERG, which operates openly as a party within a party, with its own leader, whip and policy."
It comes as at a time of heightened political tension in Britain, with less than six weeks to go before the world's fifth-largest economy is scheduled to leave the European Union.
"I am saddened by this decision — these are people who have given dedicated service to our party over many years, and I thank them for it," May said in a statement after the decision was made public.
"I am determined that under my leadership the Conservative Party will always offer the decent, moderate and patriotic politics that the people of this country deserve."