Figures such as Richard Branson and Charles Dunstone join printers and farmers in calling on people to reject Brexit on Thursday.
Remain campaigners are claiming a coup after winning the backing of 1,285 business leaders on the final day of the EU referendum campaign.
The firms, employing 1.75 million people in large, medium and small businesses, have written an open letter saying Britain's economy is better off in Europe.
They include 51 FTSE-100 companies and 910 smaller firms, from salt makers in Anglesey and dairy farmers in Devon to printers in Antrim and whisky distilleries in the Scottish Highlands.
And the top backers include Virgin boss Sir Richard Branson, Sir Charles Dunstone, of Carphone Warehouse, Jacqueline Gold, of Anne Summers, and Cobra beer baron Lord Bilimoria.
In their letter, first revealed by Sky News City Editor Mark Kleinman earlier this month, the business leaders write: "We know our firms are stronger in Europe.
"Our reasons are straightforward: businesses and their employees benefit massively from being able to trade inside the world's largest single market without barriers.
"As business people, we are always looking to the future – and a future inside the European Union is where we see more opportunities for investment, growth and new jobs."
They continue: "We know that Britain leaving the EU would mean having to re-establish terms of trade from scratch with our home market of 500 million consumers. That wouldn't just hurt exporters but the hundreds of thousands of small and medium firms who do business with them.
"Even those that want Britain to leave say that, in the short term, Brexit would lead to economic uncertainty and would put jobs at risk. Smaller businesses and the people they employ are particularly vulnerable to any economic shock which could follow a vote to leave the EU.
"Britain leaving the EU would mean uncertainty for our firms, less trade with Europe and fewer jobs. Britain remaining in the EU would mean the opposite – more certainty, more trade and more jobs.
"EU membership is good for business and good for British jobs. That's why, on June 23rd, we back Britain remaining in the EU."
Remain campaigners claim the big show of support from UK businesses puts to bed the claim of Leave campaigners that business is split on the Europe issue.
In the letter they say: "Businesses and their employees benefit massively from being able to trade inside the world's largest single market."
And they claim the Leave campaign lacks an economic plan for Britain. Leaving the EU, they say, could cause an "economic shock" that would hurt British businesses.