Shiv Sena nominee Prakash Jadhav shattered Mohite's dreams of representing Ramtek constituency as a Congress member, winning the by-poll by a comfortable margin of 32,571 votes.
Jadhav polled 2,31,239 votes, while Mohite received 1,98,668.
Mohite, who was a minister in the BJP-led government at the Centre, quit the Lok Sabha seat and the party to join the Congress a few months ago.
His resignation necessiated the April 9 by-poll in Ramtek seat spread across six assembly segments. It is one of the largest parliamentary constituencies in terms of area in Maharashtra.
The Shiv Sena had not taken kindly to Mohite's switchover to the Congress and carried out a spirited election campaign to retain the seat. Sena executive president Uddhav Thackeray had conducted roadshows, a first for him, to galvanise party cadres and mobilise voters.
Jadhav had unsuccessfully contested the 1996 parliamentary polls against then Congress nominee Datta Meghe.
Two-time state Congress president Ranjeet Deshmukh, who was also a minister in the Vilasrao Deshmukh cabinet, contested as a rebel Congress candidate and finished third with 79,638 votes.
Deshmukh, who opposed Mohite's nomination by the Congress, played spoilsport by splitting the Congress' votes and contributing to the party's defeat.