Rio, who has been the Chief Minister of Nagaland for over 11 years, tendered his resignation following his landslide victory in the just-concluded Lok Sabha polls.
Rio submitted his resignation along with that of his Council of Ministers to Governor Ashwani Kumar at Raj Bhavan.
His resignation letter read, "I hereby tender the resignation of the Council of Ministers of Nagaland government being headed by me, with immediate effect."
He was accompanied by Chief Minister designate T R Zeliang, Naga People's Front (NPF) president and Minister for Parliamentary Affairs Kuzholuzo Nienu.
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The newly elected lone Member of Lok Sabha from Nagaland also submitted his resignation from the 60-member state Assembly.
Assembly Speaker Chotisuh Sazo said, "I have accepted his resignation."
While declaring his willingness to contest the parliamentary polls, Rio had said that he wanted to go to New Delhi to play a bigger and important role for the Naga people and the North East region.
Rio defeated closest Congress rival KV Pusa by 4,00,225 votes in the recent Lok Sabha polls, which is the second highest winning margin after Narendra Modi in the country.
Zeliang is slated to stake claim to form the next Democratic Alliance of Nagaland-III ministry, sources said.
With the change of leadership, reshuffle in the DAN-III ministry is also inevitable.
DAN consists of 52 MLAs in the 60-member state Assembly.
While NPF is the major constituent of DAN with 38 members, pre-poll allies BJP and JD(U) have one MLA each.
Four NCP legislators and eight independent MLAs also joined the ruling front in 2013.