In a resignation letter seen by AFP, Nosiviwe Mapisa-Nqakula said she decided to step down with immediate effect to uphold the integrity of parliament and focus on the investigation against her.
"Given the seriousness of the much-publicised allegations against me, I cannot continue in this role," she wrote.
Mapisa-Nqakula is accused of soliciting hefty amounts in bribes from a former military contractor during her previous tenure as defence minister. She denies the allegations.
Coming just under two months before national elections, the case has added to the woes of the ANC, which is struggling in the polls amid a weak economy and accusations of graft and mismanagement.
In power since the advent of democracy in 1994, the party is expected to see its share of the vote drop below 50 percent for the first time in May, potentially forcing it to form a coalition to remain in power.
The ANC praised Mapisa-Nqakula for protecting its reputation by stepping aside before being asked to do so.
"We value her commitment to maintaining the image of our organisation," the party said.
But in her letter, the 67-year-old insisted her decision was in "no way an indication or admission of guilt."
"I maintain my innocence and am determined to restore my good reputation," she wrote.
Alleged bribes
Earlier this week, a court had rejected Mapisa-Nqakula's urgent application seeking to prevent a possible arrest.
It followed a March raid carried out by members of a top investigative team at Mapisa-Nqakula's residence, a high-end property in an eastern suburb of Johannesburg.
Shortly after, she announced she would take "special leave."
Local media reports had accused the ANC veteran of soliciting 2.3 million rand ($121,000) in bribes from a former military contractor.
Opposition parties including the radical leftist Economic Freedom Fighters and the liberal Democratic Alliance (DA) welcomed her resignation.
"This resignation is a victory for accountability and Parliament. And by extension the people of South Africa," the DA said. "Those entrusted to lead and represent South Africa must be up to the task."
Parliament said the speaker would be replaced by her deputy, Lechesa Tsenoli.
Mapisa-Nqakula served as defence minister between 2014 and 2021 before being appointed speaker in a move that drew much criticism from the opposition.
At the time, she had come under fire for perceived incompetence in responding to a spate of deadly unrest that saw more than 300 people killed.
She is the latest in a string of senior ANC politicians, including the president and vice president, to be embroiled in graft scandals.
South Africans head to the polls in national and provincial elections on May 29.
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