The Danish company said on Monday it would invest the money over 10 years in the center based at the University of Oxford, which will employ 100 scientists hunting for new ways to treat type 2 diabetes.
Britain's vote last year to leave the European Union was disappointing but did not undermine the case for working with a renowned center of science, said Mads Krogsgaard Thomsen, Novo's chief scientist.
"There's no doubt that Brexit created uncertainty for a period in our deliberations," he told Reuters.
"It is unfortunate, but we've passed that challenge and I'm convinced we've no need to worry...Oxford is a worldwide powerhouse in medicine."
The decision was welcomed by the government of Prime Minister Theresa May, who last week highlighted life sciences when she laid out a new industrial strategy designed to re-balance Britain's heavily services-based economy after it leaves the EU.
Treasury minister David Gauke said Novo's move was "a vote of confidence in the UK's position as a world-leader in science and research".