Mugabe was swept from power as his autocratic rule crumbled within days of a military takeover.
“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.
The bombshell news was delivered to a special joint session of parliament.
Lawmakers had convened to debate a motion to impeach Mugabe, who has dominated every aspect of Zimbabwean public life since independence in 1980.
It was greeted on the streets of the capital Harare with car horns and wild cheering.
It capped an unprecedented week in which the military seized control, tens of thousands of Zimbabwean citizens took to the streets to demand the president go and 93-year-old Mugabe wrestled to remain in power.
Mugabe had ruled Zimbabwe almost unopposed since the country won independence from Britain.
But his efforts to position his wife Grace as his successor triggered fury in the military that had underpinned his regime.