Every Premier League club supports Great Britain remaining in the European Union, according to the league's executive chairman Richard Scudamore.
He said opposition to the EU would be "incongruous" in the context of the league's commitment to "openness".
Speaking to BBC Radio 5 Live, Scudamore criticised the idea that a break from Brussels should be pursued purely to reclaim British sovereignty.
He told the programme: "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 co-operation.
"There is an openness about the Premier League which I think it would be completely incongruous if we were to take the opposite position."
All 20 clubs which currently compete in football's top tier are also in support of staying in Europe, Scudamore added.
The top-flight showcase generated ?3.3billion of revenue last season, according to a review by Deloitte, and boasts worldwide television contracts worth ?8.3bn.
As well as running counter to the ethos of the Premier League, the football chief 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 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 ?350m we send to Brussels each week on our priorities, like grassroots sport."