The Colchester MP Will Quince has stepped down as a Parliamentary Private Secretary because he can't support the government's Brexit deal.
Mr Quince, a ministerial aide to Defence Secretary Gavin Williamson, hit out at the backstop plan to avoid a hard border with Ireland, saying it could result in the UK being "tied to the negotiating table for years, until we agree to anything in desperation" to get out of the situation.
More than 20 of the 60 Conservative MPs in the Anglia region are now expected to vote against the EU withdrawal agreement negotiated by the government.
The crucial vote is being held in the House of Commons on Tuesday after five days of debate.
Mr Quince has been the Conservative MP for Colchester since 2015 after beating the longest-serving Liberal Democrat Sir Bob Russell.
Mr Quince had a meeting with the Prime Minister on Thursday but in his letter of resignation said it was "with deep regret" that he could not support the withdrawal deal.
"Not withstanding the huge accomplishments of the Withdrawal Agreement, due to the absence of a unilateral-exit mechanism and the consequence indefinite nature of the customs union if the backstop is triggered, it is with deep regret that I cannot support it as it stands."
Analysis by ITV News Anglia of the comments and speeches of MPs in the Anglia region suggests as many of half of the Conservative backbenchers will vote against the government deal.
Of the 60 Conservative MPs in the Anglia region, 20 are on the government payroll as ministers or whips and so would be expected to support the deal or resign.
There are 40 backbench Conservative MPs and as many as 21 have indicated that they will not support the EU deal when it comes to the vote on Tuesday.