Gerard Coyne, who stood against Len McCluskey to be leader of Unite, has been sacked via email in what he is calling a "stitch-up".
Mr Coyne, Unite's West Midlands Regional Secretary, was suspended just before the result of the election was announced in April after 16 years with the union.
He lost the election, but is challenging the result with the Certification Officer.
Mr Coyne said he was notified by email that he has been sacked from his position following a disciplinary hearing.
"I am deeply disappointed, but not surprised at my dismissal," he said."When you are in a kangaroo court, you are rarely surprised by the outcome.
"I have held the post for 16 years and no complaint was raised during the hearing about how I carried out that role.
"However, during the disciplinary process, I was informed that union rules require a regional secretary to be 'the General Secretary's representative in the region'.
"It was implied that, because of the way I criticised Len McCluskey during the campaign, I could not fulfill that role."
Mr Coyne said he faced seven charges.
He said: "Three of those charges I managed to knock out before the hearing and three were dismissed at the final hearing.
"The final one related to an alleged technical data breach, which it was claimed had damaged Unite-Labour Party relations.
"This preposterous trumped-up charge has been used to indict me - even though the returning officer from Electoral Reform Services had already ruled that there was no breach of the rules.
"It was always clear to me that the charges were nothing more than a stitch-up. My real 'crime' was having the audacity to challenge Mr McCluskey in the General Secretary election that he called unnecessarily.
"It is a public warning to any member of Unite's staff who is thinking of challenging the way the McCluskey gang run the union: step out of line and you will be out of a job. Political dissent is not tolerated inside Unite.
"I will be appealing against the decision."
A Unite spokesman said: "The decision is subject to a right of appeal to Unite's executive council, and the union will be offering no further comment on the matter."