The group - the main source of non-governmental biomedical research in the UK - said it cut its exposure to the pound to an all-time low ahead of the EU referendum, scrapping a rule to hold at least a quarter of its assets in sterling.
This helped deliver an 18.8% surge in the trust's assets under management to £20.9bn in the year to 30 September, putting it on track to invest at least £5bn into medical research over the next five years.
Its returns came as other investment firms have been battered by the pound's sharp plunge in value, with Britain's decision to quit the EU sending it tumbling to 31-year lows against the US dollar.
The fund said the global spread of its investments helped it weather the Brexit blow, as it did in the financial crisis.
Danny Truell, managing partner of Wellcome Trust's investment team, said: "The decision to reduce home country bias and to diversify assets and geographical exposure has borne fruit.
"Although future investment returns are unlikely to match recent experience, we remain confident that the portfolio should generate sufficient cash flows to insulate the trust from potentially more difficult conditions."
The trust said it saw double-digit returns from all its investment sectors, except property.
Its investment budget has doubled in the past 10 years and hit more than £1bn for the first time in the year to 30 September.
Eliza Manningham-Buller, chair of Wellcome, said: "Our charitable expenditure is now over 50% more a year than it was five years ago in 2011 and more than double that of a decade ago."
The trust holds direct investments in a number of technology giants, with stakes in Apple, Microsoft, Facebook and Chinese e-commerce group Alibaba.
Royal Dutch Shell is its biggest direct UK investment.
The organisation was set up in 1936.
It was launched after the death of pharmaceutical entrepreneur Sir Henry Wellcome, who left the entire share capital of his company to trustees charged with spending income to further human and animal health.
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