Conservative MP Stephen Phillips has quit over "irreconcilable policy differences" with the government.
The MP, who has held the Lincolnshire seat of Sleaford and North Hykeham since 2010, backed leaving the EU but has accused ministers of ignoring Parliament since the Brexit vote.
He said he was "unable properly to represent the people who elected me".
It comes as Theresa May said she was confident she would win a legal battle over her approach to Brexit talks.
On Thursday, three High Court judges ruled the government cannot officially notify the EU of its intention to leave, thus beginning formal talks, without Parliament's support.
In a series of phone calls, the prime minister told European Commission president Jean-Claude Juncker and Germany's Angela Merkel that the government believed it would win its Supreme Court appeal against the ruling and she was committed to triggering Article 50 by March 2017.
Although Mr Phillips represents a safe Conservative seat, his surprise departure increases the pressure on Mrs May's government - which has a working majority of 17.
It is not yet clear whether Mr Phillips, who won the seat last year with a majority of more than 24,000, will stand as an independent in a future by-election although this is thought to be unlikely.