"I have accepted his resignation with effect from Feb 19," Meira Kumar informed the house.
The 50-year-old Rajagopal, expelled from the Congress for opposing the formation of a Telangana state, shocked the nation when he used pepper spray during the rather tumultuous introduction of the bill in the Lok Sabha last week.
The first-of-its-kind act in parliament sent scores of members and Lok Sabha officials - and even some journalists in the media gallery - running for cover.
All parties then demanded action against the Vijayawada MP and the government said it would not oppose any decision taken by the speaker. The resignation of the industrialist-politician came after Meira Kumar asked the privileges Committee of the Lok Sabha, which has punitive powers, to go into the incident.
Rajagopal sent his resignation to the speaker Tuesday, hours after the Telangana bill was passed by the Lok Sabha.