Members of the Educational Institute of Scotland (EIS), the country's largest teaching union, have voted against moves to give local authorities more control over the amount of time they spend teaching from week to week.
They also rejected plans to water down a list of set tasks they are protected from doing, such as photocopying and patrolling the playground at break times.
Cosla, the umbrella body for councils, had wanted the changes to give councils more flexibility over how teachers spend their time, with financial cuts reducing other school staff.
However, with 41% of EIS members backing the changes and 59% opposing them after a 30% turnout, the vote has a significant impact on other negotiations between the EIS and Cosla.
Cosla officials had backed a 1% pay rise for teachers this year with a further 1% rise in 2014/15 - a pay offer which could now be taken off the table.
The body had also agreed to reverse, at least partly, a cut to the pay of short-term supply teachers which has led to chronic shortages in some parts of Scotland, but that also is now under threat.
The issue will now be discussed jointly by unions, the Scottish Government and Cosla, as well as by the Cosla executive and the EIS executive over the coming weeks.
If no agreement can be reached on the increases in salary for all teachers one possible outcome is that the EIS ballots its members on strike action.
Billy Hendry, Cosla's human resources spokesman, said he was "bitterly disappointed" with the ballot result.
"The simple fact is that, as employers, we have put together the very best components of an offer in agreement with the unions and the Scottish Government," he said.
"The offer is a package and is not open to any one of the three parties to cherry-pick the bits that they like best and reject the rest."
Michael Russell, the Education Secretary, also expressed disappointment, saying the development would have to be discussed with the Scottish Negotiating Committee for Teachers - comprising unions, the Government and Cosla - which meets today.
However, Larry Flanagan, general secretary of the EIS, said the result was a clear rejection by Scotland's teachers of the suggested changes at a time when staff were under pressure to deliver the new Curriculum for Excellence (CfE).
He said: "No-one wishes to see disruption in our schools, particularly at such a crucial phase in the implementation of CfE. The ballot result, however, is indicative of the mood of teachers who, after a two-year pay freeze, increased pension contributions and in the face of excessive workload, have simply said enough is enough."
Brian Boyd, emeritus professor of education at Strathclyde University, said it was the wrong time to be fighting over terms and conditions.
"Flexibility is something that employers are entitled to, but this is simply the wrong time to be having a face-off over the issue," he said. "The issue of supply must be addressed as a matter of urgency, but other issues should wait until after the successful delivery of the CfE."
Under the McCrone agreement in 2001, teachers were given a significant pay rise and maximum weekly teaching limits to free up protected time for marking and preparation.
However, councils argue the deal left schools with too little control over teachers' working week, particularly when there are staff shortages through absence or cuts.
They also argue positive aspects of the deal such as greater professionalism have not been delivered fully.