The director general of the British Chambers of Commerce, John Longworth, has resigned after being suspended for saying the UK's long-term prospects could be "brighter" outside the EU.
The BCC said Mr Longworth had accepted his support for leaving the EU was "likely to create confusion".
He revealed his support for "Brexit" at the BCC annual conference on Thursday.
His resignation was the "right thing to do", the boss of one of the UK's largest chambers of commerce said.
Announcing Mr Longworth's departure, the BCC said he had breached the group's official position of neutrality on the referendum.
BCC president Nora Senior said the organisation was "non-partisan" and had decided not to campaign for either side in the forthcoming UK referendum on EU membership.
She added: "John Longworth and the BCC board recognise that John's personal view on the referendum is likely to create confusion regarding the BCC's neutral stance going forward.
"In light of this, John has taken the decision to step down as director general and his resignation has been accepted by the board with effect from 6 March 2016."
She stressed that his resignation was "agreed mutually between Mr Longworth and the BCC Board, and there were no external factors involved".
James Ramsbotham, chief executive of the North East Chamber of Commerce, told BBC Radio 5 Live: "He's to be congratulated for deciding to resign because that is absolutely the right thing to do."
Mr Ramsbotham said: "We certainly all knew in the hall immediately that this was expressing personal views on a British Chambers of Commerce platform, and was going to make life extremely difficult."
However, Mr Ramsbotham said he "simply didn't believe" there had been political pressure on Mr Longworth to resign.
"I haven't had a chance to speak to John since he did it," he said. "I think he obviously feels his views very strongly."
He added: "Indeed we are becoming very aware across the country that there are a lot of people who feel very strongly on either side of this and he clearly felt he needed to express it."
At the conference, Mr Longworth said the EU referendum was a choice between the "devil and the deep blue sea".
He added that voters faced "undoubtedly a tough choice".
One option was staying in an "essentially unreformed EU", with the other being the uncertainty of leaving.
He said the very best place for the UK to be was in a reformed EU, but added: "I have come to the conclusion that the EU is incapable of meaningful reform, at least in the foreseeable future."
He said his comments had been made in a personal capacity.
His remarks and his subsequent suspension prompted a political outcry, with London mayor Boris Johnson and former defence secretary Liam Fox, both prominent campaigners for the UK to leave the EU, weighing in on his behalf.
Mr Johnson called Mr Longworth's treatment "scandalous", while Mr Fox said ministers should clarify "if they were involved in any way in putting pressure on" the BCC to suspend Mr Longworth.
Who is John Longworth?
- Appointed BCC director general in 2011
- Has worked for big names, including Tesco and Asda
- Has held non-executive director positions at the Co-operative Food Group and drinks company Nichols
- Has been on the Competition Commission panel
- Has served as a consultant to Barts Hospital Trust
- Founded SVA, a science research firm
Downing Street denied any pressure was put on the BCC to suspend its director general.
British voters will be asked on 23 June whether the UK should remain a member of the EU.
The BCC, which represents thousands of large, medium and small businesses, has said it will not campaign for either side in the referendum as its membership is split.
Last month, an online poll of its members found 59.5% of more than 2,000 who responded preferred to remain in the EU and 30% said they would vote to leave.