The organization said that John Longworth has decided to step down because his "personal view on the referendum is likely to create confusion."
The group, which represents hundreds of businesses, says it will not campaign on either side in Britain's EU membership referendum, to be held June 23. A majority of its member businesses supported staying in the bloc when surveyed last year.
But last week Longworth said that "the UK would be better off taking a decision to leave" the EU.
Supporters of EU withdrawal accused David Cameron's 10 Downing St. office of involvement in Longworth's ouster. But Downing St. said Monday that "no pressure" had been put on the organization, the Associated Press reported.
Longworth said he had not been pressured to resign.
"I've voluntarily resigned as director general of the British Chambers of Commerce in order to have the freedom to express myself on the European referendum," he said.