The chairman of Japanese giant Hitachi was warned the "cold economic reality of Brexit" would mean jobs being lost in Wearside and investment being held back from the city.
The sprawling business empire has invested £50million in Wearside since 2012, most recently in new warehousing facilities opened by its subsidiary company Vantec at the Turbine Business Park in April.
Hitachi also has a train manufacturing plant in Newton Aycliffe, as well as its global rail HQ in London, said Brexit represented a "boulder on the line".
Its chairman Hiroaki Nakanishi said the UK was attractive as the "best base" for accessing the European Union's market of 500 million consumers - but warned Brexit represented a "boulder on the line" and warned of the impact leaving the RU would have on Japanese investors.
He said: "In the 80s Nissan and Toyota came to the UK on the basis that if they produced here and employed a British workforce they would be treated as European companies.
"This was only possible because Britain was inside the EU; and so the UK car industry was revived and became an exporter again.
"From Japan, this incredible success story looks like a huge gain from the UK's membership of the EU."
Mr Nakanishi made the statements in an opinion piece written for the Daily Mirror.
He went on: "We worry because those advocating Brexit have no answer to how the UK could negotiate cost-free access to this huge market from a position outside it.
"It would take a long time and result in uncertain market conditions; during this renegotiation period, investors would probably be waiting to see the outcomes, hold back on investment, and jobs would be lost.
"This is the cold economic reality of Brexit."
He continued: "Brexit would force us and similar companies to rethink, because we still have a European vision, and would be disadvantaged in pursuing it from the UK.
"We understand the EU is not perfect, but we hope the UK can continue from inside to change it for the better.
"Strong national policy and membership of Europe can go together.
"From our viewpoint, attracting foreign investment and high-value job creation depends on staying in and continuing to create a competitive internal market, and in this we hope the UK will be the leading force it has always been."
Unions said Mr Nakanishi's remarks were a strong warning to voters, and again urged members to cast their ballots in favour of EU membership on June 23.
Northern TUC spokesperson Neil Foster said: “The comments from Hitachi's Chairman are a stark reminder of what is at stake in this referendum for the North East and our economic future.
“If we stay in the EU, our region can attract more investment, create high-quality jobs and support manufacturing. But if we leave the EU we risk losing rights at work, losing investment and losing good quality jobs in the years to come.
"The Northern TUC is proud to have been a driving force in the campaign for the Intercity Express Programme, and for Hitachi to come to the North East. But Brexit will put hundreds of the jobs we gained at risk, as well as thousands more down the supply chain.
He added: “We want the best quality jobs for today's workers and the next generation in the North East – and that's we should remain in the EU and build on the success of Hitachi's arrival."