Dragons' Den stars said they would not back the "reckless risk" of leaving the EU as the Remain camp claimed 1.2 million smaller firms would be directly affected.
A group of seven past and present members of the TV show's panel of leading entrepreneurs said they were "in" because Brexit posed a "serious risk" to the UK economy.
Business Secretary Sajid Javid will appear with one - Piers Linney - as well as Cambridge Satchel Company founder Julie Deane to press the case at an event in Birmingham.
Mr Javid - who has been accused by former cabinet colleague Iain Duncan Smith of privately backing divorce from Brussels - will use the event to warn of the impact on SMEs.
He will cite analysis by the Department for Business, Innovation and Skills (BIS) estimating that 8% export to the EU and a further 15% are in the supply chains of other businesses that export to the EU.
Britain Stronger in Europe said that amounted to around 1.2 million firms if you include the smallest businesses, including non-employers, which don't have to register to pay VAT.
BIS qualifies its estimate of what proportion are directly or indirectly involved in EU exports as "indicative estimates" as it uses data whose definitions "do not entirely align".
Mr Javid said: "This shows yet again that Britain's small businesses are stronger, safer and better off in Europe.
"If we leave the EU, small firms are on the front line and that's a gamble with people's livelihoods I'm not willing to take.
"Small businesses are the backbone of our economy. Let's not break that backbone with a leap into the dark."
The statement from the Dragons was signed by James Caan, Hilary Devey, Kelly Hoppen, Touker Suleyman, Sarah Willingham, Simon Woodroffe and Mr Linney.
It said: "Leaving the EU constitutes a serious risk - to the British economy as a whole and to millions of businesses, including start-ups, small firms and larger corporations.
"We only invest when an opportunity seems right and risks are understood and manageable, so we are used to saying "I'm out" most of the time.
"But on the issue of the UK's membership of the EU, our answer is clear: we're In".
Ms Deane said: "I know from my own experience that when there is uncertainty and the times get worse, small businesses including the self-employed are hit first. People don't buy as much and it's harder to get money from investors.
"So for me there is no doubt: small businesses, start-ups and the wider economy are far better off inside the EU".
John Longworth, former director general of the British Chambers of Commerce who now chairs the Vote Leave Business Council, said: "I agree with what Sajid Javid used to say about the EU - before he changed his mind for reasons we can only guess at.
"EU rules damage all British businesses, and smaller businesses in particular - and hold us back from trading freely with the rest of the world.
'These Government figures are extremely questionable. The reality is that only 6% of British firms export to the EU, but 100% are caught up in red tape and costs from Brussels. If we Vote Leave and take back control of our economy, our businesses will thrive."
'I hope that Sajid Javid's priority is not his political career - rather than helping British businesses.'