"I Robert Gabriel Mugabe in terms of section 96 of the constitution of Zimbabwe hereby formally tender my resignation... with immediate effect," said speaker Mudenda, reading the letter.
His resignation follows a debate on impeachment proceedings against Mugabe, the country's sole leader since independence 37 years ago.
As the 93-year-old autocrat faced intensifying pressure to quit, southern Africa's regional bloc announced it was dispatching the presidents of Angola and South Africa to Harare to discuss the crisis.
Lawmakers began the historic impeachment debate shortly after ousted vice president Emmerson Mnangagwa, who could be the country's next leader, told the 93-year-old Mugabe to step down.
Mnangagwa, formerly one of Mugabe's closest allies, said in a statement that Zimbabweans had "clearly demonstrated without violence their insatiable desire" for Mugabe to resign.
"It is my appeal to President Mugabe that he should take heed of this clarion call," he said.
Parliament speaker Jacob Mubenda authorised a joint session of the House of Assembly and the Senate to debate a motion to impeach the man who is the only leader most Zimbabweans have ever known.
"This motion is unprecedented in the history of post-independence Zimbabwe," he declared before the decision.
There were tense protests outside parliament as hundreds of demonstrators -- from rival political parties -- shouted for Mugabe to go.
Activists hung brightly coloured postcards with political demands on lines strung between trees in a park near to parliament.
"Rename Robert Mugabe Road" said one, "free elections, no to police brutality" said another.
Morgan Tsvangirai, leader of the main opposition Movement for Democratic Change, said between the sessions that his party supported impeaching the president.
A bubbling factional squabble over the presidential succession erupted two weeks ago when Mugabe fired Mnangagwa.
The dismissal put Mugabe's wife Grace in prime position to succeed her ageing husband, prompting the military to step in to block her path to the presidency.
After Mnangagwa fled abroad, the army took over the country and placed Mugabe under house arrest -- provoking amazement and delight among many Zimbabweans as his autocratic reign appeared close to an end.