Jo Stevens is the latest MP to quit Jeremy Corbyn's top team after the Labour leader said he would impose a three-line-whip on the Article 50 vote.
Stevens, the MP for Cardiff Central since last May, resigned as shadow Welsh secretary just a day after Tulip Siddiq, the former shadow early years education minister, also quit Labour's frontbench.
"I expect this to be the most important vote I will ever cast as an MP and for me it is a clear issue of principle and conscience," Stevens said in her resignation letter.
The move is another blow to Corbyn, who has promised not to block the UK's divorce from the EU despite the majority of Labour MPs backing Remain.
The Labour leader unveiled numerous amendments to the government's Article 50 bill on Thursday night (26 January), including an anti-tax haven amendment.
"Labour will seek to amend the Article 50 bill to prevent the government using Brexit to turn Britain into a bargain basement tax haven off the coast of Europe. Our country can do much better than that," Corbyn said.
"We respect the will of the British people, but not the will of this Tory government to impose fewer rights at work and worse public services, while the largest corporations pay even less tax.
"Labour will ensure that the British people, through parliament, have genuine accountability and oversight over the Brexit negotiations because no one voted to give May a free hand over our future."
The EU Notification of Withdrawal was tabled before parliament on Thursday, with a second reading debate scheduled for Tuesday 31 January and Wednesday 1 February.
A further three days of debate will be held from Monday 6 February, including the draft law going through its third reading and committee stage, with a final vote being taken on Wednesday 8 February.
The draft legislation comes after the Supreme Court's landmark ruling on Tuesday, which saw the top judges decide eight to three that MPs and peers should have a vote on invoking Article 50.
The decision could scupper May's plan to trigger Brexit talks by the end of March since the House of Lords, with more than 300 Labour and Liberal Democrat peers, could delay the process of the bill.