"I am not paying that bill on the 1 December, if people think I am they have got another thing coming. We are not suddenly going to get our check book out for 2 billion euros. The way this was presented is an unacceptable way to behave," he said.
Read MoreBritain's growth slows as row over EU bill flares
The U.K. will have to pay a top-up payment on the 1 December after calculations carried out by EU statistics agency Eurostat revealed a gap between what Britain has been paying and what it should be paying, when accounting for its economic recovery.
Countries such as France and Germany will likely receive rebates from the EU as their economies have struggled to deliver convincing growth.
Cameron said it was a "deep frustration" that certain European economies were not growing faster and that slow growth from its neighbors in the EU was a "threat" to the British economy.
Read MoreUK retail sales dented by warm weather
The prime minister said there was "downright anger" in response to the "sudden production of the bill".
"It is appalling. We are a leading player in this organisation," he added.
Speaking on whether the bill could jeopardize the U.K's membership of the EU, the prime minister said "it certainly doesn't help."
Europe's finance ministers are to meet at an emergency meeting following the demands from some countries, including Italy and the Netherlands, to contribute billions more to the EU budget.
Earlier on Friday, other European politicians attending the European Commission meeting in Brussels remained tight lipped on the EU's budgetary recalculations.
President of the Eurogroup and minister for finance of the Netherlands, Jeroen Dijsselbloem said the new governments in France and Italy appeared "quite ambitious in terms of reforms and modernizing their society".
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"Some reforms are being pushed though in Paris and in Rome and I think that is crucial – so let's talk about that today," he told CNBC
"We have to step up our efforts certainly on the structural reform side, which makes Europe more competitive. Member states have work to do," he added.
President of Lithuania Dalia Grybauskaitė said the EU's new calculations were simply based on the prognosis of each country's economy.
"Each year we have final revision of our budgets because the payments that we pay as a member of the EU are based on prognosis, and by the end of the year the European Commission revises - some countries need to pay and some countries receive back," she said.
When asked if this was a message to David Cameron she said, "Probably, yes."