The Movement for Democratic Change (MDC) sacked Elton Mangoma, the party's deputy treasurer and a former energy minister, for "defying" the party.
The MDC's top decision-making body "unanimously" resolved to dismiss Mangoma "for undermining the authority of the party," spokesman Douglas Mwonzora told AFP.
Mangoma can appeal the decision to the same body.
He was suspended last month after he suggested Tsvangirai should step down as leader of the MDC following last year's election defeat to veteran ruler Robert Mugabe.
Mangoma had sought to appeal his suspension in court, in a case that has yet to be heard.
His call sparked furious disagreement within the party that degenerated into physical attacks, with suspected Tsvangirai loyalists beating Mangoma in front of the party's headquarters.
The party's treasurer Roy Bennett, who is exiled in South Africa, and other officials have made similar calls for Tsvangirai to resign after losing to Mugabe in the July 31 general elections.
The MDC, formed in 1999 by an alliance of trade unions and civic groups, has been seen as the most credible challenger to Mugabe's nearly 34-year hold on power.