Sunderland AFC has joined with Middlesbrough and the 18 other Premier League clubs in urging voters to cast their ballots in favour of remaining in the EU.
Richard Scudamore, the executive chairman of the Premier League, said all 20 clubs which currently compete in football's top tier were in support of staying in the European Union.
Mr Scudamore, one of football's most senior figures, said the League was committed to "openness" and leaving the EU would be damaging to the English game.
Speaking to BBC Radio 5 live, Mr Scudamore also criticised the idea that an break from Brussels should be pursued purely to reclaim British sovereignty.
"That doesn't seem to sit very well when you travel the world like we do being welcomed because of the fact that we are open for business, open for discussion, and open for cooperation," he said.
"There is an openness about the Premier League which I think it would be completely incongruous if we were to take the opposite position."
Football's top-flight showcase generated £3.3billion of revenue last season, according to a review by Deloitte, and boasts worldwide television contracts worth £8.3billion.
As well as running counter to the ethos of the Premier League, Mr Scudamore said Britain risked losing respect across the globe if it left the EU.
He said: "We would just be, I think, respected less around the world for not wanting to be part of something.
"Nobody bears the scars more than me of having to go and negotiate in Brussels and try and organise things a little bit in our interests in terms of the European machine."
He added: "Ultimately you can't break away, you can't just pull out, you have to get in and negotiate and try and organise and try and influence."
But the Leave camp have criticised Mr Scudamore's assessment, saying Brexit could shore up more funds to be invested in grassroots sports.
Vote Leave's Robert Oxley said: "EU rules hurt both our ability to develop home grown talent and restrict access to the global talent pool.
"That's the worst of both worlds for football fans and the teams they support. If we take back control, we can spend the £350million we send to Brussels each week on our priorities, like grassroots sport."