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EU referendum: 250 business leaders back exit, say campaigners
But Matthew Elliott, Vote Leave's chief executive, said it had a "growing list of business supporters".
Vote Leave also announced that its Business Council would be chaired by John Longworth, who resigned his role as director general of the British Chambers of Commerce (BCC) earlier this month.
By Joe Lynam, BBC business correspondent
The pro-Brexit camp has striven to counter the impression that most big companies prefer to remain in the EU. A third of FTSE 100 bosses publicly supported staying in last month.
This business council, under the auspices of Vote Leave, tries to address that by bringing together entrepreneurs and former company leaders who all want Britain to quit the EU.
There are some colourful names and a few well-known bosses including the former HSBC boss Michael Geoghegan, Tim Martin from JD Wetherspoon and Phones 4u founder John Caudwell.
Many, like Sir Stuart Wheeler and co-founder of Carphone Warehouse David Ross, have ties to UKIP and the Conservatives.
But there are no current FTSE 100 chief executives or chairmen and only 13 out of the 250 signatories are women - including Sir Rocco Forte's sister Olga and his niece Alex Polizzi.
Mr Longworth's resignation followed his suspension for saying the UK's long-term prospects could be "brighter" outside the EU when the BCC had an official position of neutrality on the referendum.
Vote Leave's Business Council will make the case that EU membership is holding back British business.
Mr Longworth said: "If we Vote Leave, liberated from the shackles of EU membership, jobs will be safer, Britain will be able to spend our money on our priorities and we can look forward to faster growth and greater prosperity in the future."
Mr Elliott said: "Vote Leave will make that case, that whilst the EU might be good for big multinationals, for smaller businesses it acts as a job destruction regulatory machine.
"Brussels hinders smaller businesses, particularly those firms who can't afford to lobby Brussels to curry favour.
"Jobs, wages and our economy will thrive when we take back control and Vote Leave."
But Business Secretary Sajid Javid, who is supporting the Britain Stronger in Europe campaign, said businesses could see "we are stronger, safer and better-off in a reformed EU".
"Survey after survey shows that small businesses - the backbone of our economy - want to stay inside the EU rather than take a leap in the dark," he said.
Sir Mike Rake, chairman of BT and fellow supporter of the In campaign, said: "Our best course is to remain in the EU, constructively engaging to improve its competitiveness."
Some of the business leaders who have signed up as backers of the Vote Leave campaign:
Emma Pullen, MD of the British Hovercraft Company
John Hoerner, former chief executive officer, Tesco Clothing UK and Central Europe
Luke Johnson, chairman of Patisserie Valerie
Jon Moulton, founder of Better Capital LLP
Rupert Hambro, director of Theo Fennell Plc
Pasha Khandaker, president of the Bangladesh Caterers Association