Some 33,000 nurses trained in the EU are registered to work in the UK, figures show.
Nursing and Midwifery Council (NMC) data, obtained by the Royal College of Nursing (RCN), shows there are more EU-trained nurses registered to work in the UK than the number of nurses employed in the whole of Wales.
More than 9,000 EU nurses joined the NMC register in 2015/16, a 21% increase on the year before.
The RCN said the Government must secure the futures of EU nurses. It comes after Simon Stevens, chief executive of NHS England, called on ministers to tell foreign NHS staff their jobs are safe post-Brexit.
He said the NHS will continue to rely on professionals from abroad in spite of initiatives to increase the number of NHS workers from within the UK.
Earlier this week, a report from MPs on the Commons Health Committee noted how poor historical workforce planning means the NHS has had too few homegrown nurses in training, while fewer have returned to the NHS after a break than predicted.
Janet Davies, chief executive of the RCN, said: "These are uncertain times for safe staffing in the health service, and a lack of concrete assurances over the future of EU nursing staff working in the UK is making the situation worse. It is vital that valued colleagues are supported to stay.
"A sustained lack of investment in training new nurses and years of pay restraint mean many experienced nurses can't afford to stay in the profession. Plans to change student funding and question marks over our future relationship with the EU place even greater pressure on the NHS.
"The Government must act now and develop a coherent and sustainable workforce strategy for the future that recognises the critical contribution of overseas nurses as well as the pressing need to educate, recruit and retain a homegrown nursing workforce.
"Allowing this ambiguity about our NHS workforce to continue is a completely unfair way of treating people who are caring for our friends and families every single day. It may also prompt many to leave the UK, making it even harder for the NHS to provide safe patient care."
The House of Lords is due to debate the impact of the vote to leave the EU on safe staffing levels in the NHS.
Dr Mark Holland, president of the Society for Acute Medicine, said: "If it were not for health professionals from overseas, we would probably be unable to run any service - let alone a safe service.
"We fully support all colleagues who trained overseas remaining in the UK as, without them, we will be unable to provide services.
"As a nation, we must ask ourselves why we struggle to train and retain an adequate workforce in the NHS. The real issue here is not Brexit but our inability to train and retain enough healthcare workers."
As of March 31, there were 659,303 nurses and midwives on the NMC register. Of these, 33,248 were EU-trained, which is 5% of the workforce.
A Department of Health spokesman said: "NHS staff - including those from overseas - make a huge contribution to our country.
"The Government has been very clear that when we leave the EU, we fully expect that the legal status of EU nationals living in the UK, and that of UK nationals in EU member states, will be properly protected.
"Making sure we have enough nurses is a top priority for this Government. There are already 11,000 more nurses on our wards since 2010 and 50,000 nurses currently in training - with thousands more training places for home-grown nurses being created to deliver a safer, seven-day NHS."
Copyright (c) Press Association Ltd. 2016, All Rights Reserved. Picture (c) Peter Byrne / PA Wire.