The letter is signed by 282 influential names working across film, music, theatre and literature, including Benedict Cumberbatch, Helena Bonham Carter and Patrick Stewart.
The signatories stress the "important" role the EU has played in funding and promoting the UK's creative industries across the globe.
The letter says, "From the smallest gallery to the biggest blockbuster, many of us have worked on projects that would never have happened without vital EU funding or by collaborating across border.
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"From the Bard to Bowie, British creativity inspires and influences the rest of the world. We believe that being part of the EU bolsters Britain's leading role on the world stage."
The EU, it points out, will invest €1.4bn across British creative and cultural sectors between 2014 and 2020 through its Creative Europe programme.
More than half of this budget is allocated to the media strand which supports film, television, new media and the growing video games sector.
Notable projects to receive EU funding include the British-made film Slumdog Millionaire, directed by Danny Boyle, another signatory of the letter. The project secured €1.3m to help the distribution of the Oscar-winning film.
The British made global box-office hit The King's Speech was also awarded more than €1m from the EU programme.
European Regional Development Fund (ERDF) continues to make capital investment available to UK arts organisations operating outside London.
The letter says the Liverpool Everyman and The Sage at Gateshead have each received considerable ERDF awards to support their redevelopment and enhance the cultural offering available to local communities.
Many education and outreach programmes also benefit from access to the €80bn innovation fund, Horizon 2020, it points out.
Some 56 per cent of the UK's cultural sector exports go directly to the EU.
Prominent creative business leaders like Universal Music UK (representing the late Amy Winehouse, Elton John and Sam Smith), Working Title Films (whose productions include Les Misérables and the Theory of Everything) and Penguin Random House (50 Shades of Grey) have also signed the letter.
Publication of the letter has been welcomed by UK MEP Julie Ward, the Labour party spokesperson for culture and education.
She said the industry was sending out a clear message: "It needs Britain to remain in the EU to shape future trade deals and ensure the digital single market strategy works in the best interest of British creators and consumers alike."
Responding to the letter, she said, "The creative sector has added yet another compelling voice to the economic argument for a vote for Britain to remain in the EU.
"But more importantly, the cultural sector has reminded all of us that this referendum is about so much more than economics. Culture and the arts have always played an important role in the European project, bringing people together across borders to celebrate humanity, explore the more complex questions we each face in life and promote health and wellbeing."
The letter came out in the same week that the Creative Industries Federation (CIF) revealed that 96 per cent of its industry members wanted Britain to remain in the EU and 300 prominent historians sent a letter to the Guardian to remind readers about the influential role the UK plays in Europe.
Meanwhile, a survey at the weekend found 88 per cent of 600 experts fear a long-term fall in GDP if UK leaves single market.
Reacting to the findings of the poll by the Observer newspaper, UK MP Alan Johnson, who chairs the Labour In for Britain campaign, said, "Another day, another set of economic experts adding their authoritative voices to the argument to stay in the EU. The economic evidence isn't so much piling up as becoming a landslide: leaving Europe would hurt Britain's economy. We need to remain in Europe for Britain's jobs, growth and prosperity."