Support staff, who say they are being "pushed and bullied" into teaching whole classes, are poised to vote to pull out of the workforce agreement.
Unison's continued participation in the reforms rests on a knife-edge as the public-service union meets for its annual conference in Bournemouth on Monday.
Delegates, who represent most support staff, will discuss motions calling for the union to renegotiate the agreement or suspend its involvement altogether.
Unison's leaders want to stay on board but fear defeat is a real prospect.
Christina McAnea, Unison head of education, said: "It is very close. It is going to be a very tough debate and I am not confident of the outcome this year."
Withdrawal would represent a huge blow to one of the Government's flagship education polices.
David Miliband, school standards minister, tried to head off opposition at a meeting of local Unison officials in London earlier this month. But he and Dave Prentis, Unison general secretary, are understood to have faced largely hostile questioning.
Grassroots anger at the failure of the agreement to deliver a better deal for support staff is clear in the wording of some conference motions.
These claim the deal has been "largely negative and counterproductive", that heads and governors in many areas are "driving a coach and horses" through it, and threaten industrial action if the Government fails to negotiate national terms and conditions for support staff.
Alex Knutsen, Unison Brighton secretary and one of the main opponents, said a survey of 115 English Unison branches had uncovered widespread discontent.
Of 70 responses, 65 said support staff were not paid enough, 56 backed suspension of the deal and 41 thought the status of support staff was lower because of the agreement. Last year Unison narrowly voted to remain party to the agreement, with a 60 to 40 per cent split. Those backing the deal which did not come into force until September said that it should be given time to work.
It was signed in January 2003 by unions, the Government and employers and was designed to reduce teacher workload and improve support staff pay and conditions.
Ms McAnea said the deal was starting to deliver benefits and that 20 per cent of local authorities had negotiated new support staff career structures that had led to pay rises of 9 to 10 per cent.
But she admitted progress was slow and that two-and-a-half terms into the agreement nearly half of councils had not begun to review assistants' pay.
She also fears that those calling for withdrawal are better organised than they were last year.
With the National Association of Head Teachers already threatening withdrawal over funding and the National Union of Teachers shunning the deal from the start, a Unison pull-out could leave the Government trying to implement workforce reforms without the co-operation of the biggest classroom teacher, head and support staff unions.
Many London Unison branches have reached local understandings with the NUT to defy the deal and stop support staff taking whole classes.
The NUT continued its campaign against the agreement this week by writing to every school in England and Wales asking them to adopt the same policy.