Tycoon Sir Terry Matthews, JoJo Mamam Bebe founder Laura Tenison and Cardiff Airport chairman Roger Lewis are among 10 who have signed a letter backing the case for remaining in the EU.
It highlights access to the European market and EU investment in skills.
The Leave campaign argues that business would still be able to trade with the EU after Brexit.
It also believes funding that comes from the EU to Wales could be replaced by Westminster from the savings of not paying for EU membership.
But in the letter, the business leaders argue: "Our membership of the EU is vital to the Welsh economy - giving Wales favourable access to a market of 500 million people, supporting competitiveness and innovation and spreading investment in infrastructure to all parts of Wales."
At the moment, 41% of exports from Wales go to the European Union.
That has risen from £4.7 billion worth of goods and services in 1999 to £5 billion in 2015.
Those who support leaving the EU claim it would allow exporters to focus more on growing markets further afield.
Exports from Wales to countries outside the EU have risen from £1.7 billion in 1999 to £7.2 billion  in 2015.
If Brexit does happen, selling abroad will be dependent on what trade deals can be negotiated with other countries.
Skills is one of the key areas for businesses and the letter goes on to say: "Investing in skills is so important to raise income levels and encourage further business growth, and in this decade, we will see £1 billion of EU funds invested in young people, training and skills."
Others who signed the letter include:
Advocates of leaving the EU argue the money saved from membership fees could be reinvested in other areas.
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The independent think-tank, Institute for Fiscal Studies (IFS), has warned the savings from membership fees do not take any account of the potential damage to the economy of leaving the EU.
Most business surveys suggest that firms are, to a greater or lesser extent, in favour of remaining in the EU.
In April, aerospace company Airbus, wrote to its 6,000 workers in Flintshire warning about the risks of voting to leave the European Union.

Massey, 22, has signed until 2017, while 22-year-old Eastman has agreed a deal until 2016 and Kent, 18, will stay with the League One side until 2018.
All three have featured prominently this term, with teenager Kent making his first senior start in August.
"It's been a long journey to get to where I am and I can't thank everyone enough," he told BBC Essex.
"I'm feeling comfortable in the team and I'm getting used to my surroundings.
"Once I started playing a few games I think the club wanted to reward me. I couldn't turn it down - I love playing here."
Young U's midfielder Sammie Smzodics also signed a contract until 2018 last week.
The Welsh economy is "stronger, safer and better off" in the European Union, a group of top business figures says.
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Colchester United have handed new deals to striker Gavin Massey and defenders Tom Eastman and Frankie Kent.