The spokeswoman for the ruling Pakistan People's Party resigned on Saturday days after backing the claims of diplomatic immunity by a US gunman who killed two Pakistanis last month.
Pakistan's ties with the United States have been strained since police arrested Raymond Davis, who confessed to killing two men in self-defence on a busy street in the eastern city of Lahore on January 27.
On Monday Fauzia Wahab said diplomats have immunity and that Davis had an official visa, in comments swifty dismissed as personal views by a presidential spokesman.
"I have resigned because I gave that statement in my personal capacity," Fauzia Wahab told AFP.
"I do not want to appear before Lahore High Court as an office-bearer of the Pakistan People's Party. To uphold the dignity and respect of my party I have resigned from my post," she added.
Wahab had said that Davis had an official business visa "so why argue and why are we risking our overall good reputation before the rest of the world?" "We have always abided by international laws and conventions," she had said.
The ruling party has also ditched Pakistan's former foreign minister Shah Mehmood Qureshi in a recent cabinet reshuffle.
Qureshi, who was still in his post at the time of the shootings, said Wednesday that in his view Davis did not have full diplomatic immunity.
Pakistan's unpopular President Asif Ali Zardari and Prime Minister Yousuf Raza Gilani, struggling to keep his coalition in power, say it is up to the courts to decide on the case.
On Wednesday, Gilani asked Islamic scholars to help, suggesting that the families might pardon the American and telling clerics that the government was caught between a public backlash and international anger.
US Senator John Kerry visited the country this week to hold talks with Pakistani leaders aimed at resolving a bitter diplomatic row.