The Stormont Assembly faces a crucial vote
Pauline Armitage told BBC Radio Ulster's Talkback programme on Thursday she could not vote for a policy which she felt was destroying the union.
She said she had grave doubts about IRA decommissioning and the direction of the peace process.
The development comes 24 hours ahead of the crucial assembly vote.
Mr Trimble needs the votes of all his UUP colleagues, as well as other pro-Good Friday Agreement unionists to be safely returned as first minister.
Meanwhile, the Women's Coalition has said it is prepared to re-designate one of its two members as a unionist to ensure Mr Trimble succeeds.
Mrs Armitage said she did not have any scores to settle with Mr Trimble, but would be voting against Ulster Unionist Party policy.
"We are told to leave decommissioning to de Chastelain," she said.
"I think we are really leaving decommissioning to the IRA. They are calling the tune."
Sinn Fein have called for fresh assembly elections if the devolved government fails to fill the top two assembly posts.
Disarmed
Another Ulster Unionist hardliner, Peter Weir, has challenged Sinn Fein's Martin McGuinness to publicly answer questions on IRA decommissioning.
Peter Weir was speaking a day after Mr McGuinness offered to meet him to address his concerns over the IRA's move to put some arms beyond use.
On Thursday, Mr Weir said he had no intention of meeting Mr McGuinness but called on the Sinn Fein education minister to reveal details of the weapons which have been disarmed.
He added: "My policy has not changed: I do not talk to IRA-Sinn Fein."
Mr Weir said people wanted to know when the next act of decommissioning would occur and when the process would be completed.
The first and deputy first ministers will have to gain a majority of support within both the unionist and nationalist blocs in the assembly to be elected on Friday.
Mr Weir and Mrs Armitage met the head of the Independent International Commission on Decommissioning in Belfast on Wednesday.
Afterwards, both described their talks as useful.
Meanwhile, Martin McGuinness has said the suspension of the institutions would be "totally unacceptable" if the first and deputy first ministers failed to be elected.
He said suspension was a "waste of time" and that an election had to be called under the terms of the Good Friday Agreement.
Elsewhere, the Progressive Unionist Party has decided to back Mr Trimble. David Ervine said on Thursday the assembly was the only way forward.
Mr Trimble resigned in July because of an absence of IRA decommissioning and then withdrew his ministers from the executive two weeks ago to put further pressure on republicans.
Succeeded
Following the IRA move last week, he decided to bring his party back into power-sharing, and his decision has been backed by his party executive.
However, hardliners in the party have collected the signatures necessary to call an extraordinary meeting of the Ulster Unionist Council within three weeks.
Meanwhile, leader elect of the nationalist SDLP, Mark Durkan, who is hoping to win the assembly's backing to take the deputy first minister's post said all Ulster Unionists must support Mr Trimble at the election.
Alliance leader David Ford has said his party would not consider re-designating as unionists in order to secure Mr Trimble's re-election.