Nikki Da Costa, director of Legislative affairs at Downing Street has handed in her resignation.
She told Guido Fawkes: “It has been an honour to serve the PM and I have huge respect for her. This decision was very hard.”
It comes amid a wave of resignations on Thursday in response to Theresa May’s Brexit proposals.
Ms da Costa was brought in last year in the new role of Director of Legislative affairs.
The role aimed to help cope with the difficulties of pushing through legislation without a majority in the commons with a focus on Brexit and domestic bills.
She previously worked for her own company that provided business training to public affairs professionals.
Earlier on Thursday, Esther McVey, Dominic Raab and Shailesh Vara announced their resignations from the cabinet.
Northern Ireland minister Shailesh Vara went first saying that he “cannot support” the agreement between Britain and the EU.
Mr Raab resigned over the Brexit deal he negotiated, saying: "No democratic nation has ever signed up to be bound by such an extensive regime."
Remain-backing Conservative MP Anna Soubry tweeted: "Raab's resignation marks the end of PMs Withdrawal Agreement. This is v serious the PM will clearly be considering her position. My own view is that we need a Govt of National Unity and we need it now."
Later on, Esther McVey resigned. Writing in a letter to Theresa May, she said: “The deal you put before the cabinet yesterday does not honour the result of the referendum. Indeed, it doesn’t meet the tests you set from the outset of your premiership.”
