Top bosses including Sir Richard Branson, the Premier League chair and car industry executives have backed Remain ahead of Thursday's EU vote.
Premier League chair Richard Scudamore said the 20 clubs in the top tier wanted to remain and that leaving would be "incongruous" in the context of the league's commitment to "openness".
Meanwhile, Sir Richard warned a British exit from the EU would be "devastating" for the UK's long-term prosperity.
Vote Leave rejected the comments.
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It argued a British exit from the European Union would boost industry.
John Longworth, chair of Vote Leave business council, said the UK would be better off outside the EU.
"The single market isn't a nirvana, it's a mirage. The single market's a protectionist area.
"[Under Brexit] We'd be able to remove the external barriers, reduce the cost of clothing and footwear. Reduce the cost of food products we can't produce in the UK because at the moment the EU puts tariffs up to the rest of the world which we have to pay for," he told the BBC's Today programme.
But the car industry trade body, the Society of Motor Manufacturers and Traders' (SMMT), warned leaving the EU would increase costs and threaten jobs.
"Remaining will allow the UK to retain the influence on which the unique and successful UK automotive sector depends," said chief executive Mike Hawkes.
Directors at Toyota UK, Vauxhall, Jaguar Land Rover and BMW, as well as from component makers GKN and Magal Engineering, also voiced their support.
Rory Harvey, chairman of Vauxhall, said: "We are part of a fully integrated European company where we benefit from the free movement of goods and people. We believe not to be part of the EU would be undesirable for our business and the sector as a whole."
'Saddened'
Meanwhile, Premier League boss Mr Scudamore told BBC Radio 5 live that leaving would be "incongruous" in the context of the league's commitment to "openness".
"There is an openness about the Premier League which I think it would be completely incongruous if we were to take the opposite position," he said.
Sir Richard Branson, who has long backed the Remain campaign, wrote an open letter, recalling "how difficult it was" for businesses to operate effectively before the EU, adding he was "saddened" at the prospect of returning to those days.
"Although I've been living in the British Virgin Islands for some time now, I have never stopped caring passionately about the UK and its great people. I am one of the few business people who can remember how difficult it was before the EU was formed," the Virgin Group boss wrote.
And drinks giant Diageo's chief executive Ivan Menezes wrote to the company's 4,773 UK employees, telling them that it would be "better for the UK, better for Diageo and better for the Scotch whisky industry that we remain in".
Mr Menezes said Diageo benefited from ease of access to the European single market, as well as trade deals that the EU had negotiated with the rest of the world.
"The EU has so far concluded, or is negotiating, over 50 of these global agreements, many of which provide significant commercial benefits for Diageo," Mr Menezes said in his letter. Negotiating new deals after a Brexit could take years, he said.
