The open letter signed by Democratic and Republican former secretaries echoed warnings by British Prime Minister David Cameron that leaving would be a "risky bet on the country's economic future".
"A strong Britain, inside the European Union, remains the best hope in our view for securing Britain's future, creating a more prosperous Europe and protecting a healthy and resilient global economy," they said.
The letter, published in The Times newspaper, was signed by Timothy Geithner, Henry Paulson, John Snow, Paul O'Neill, Larry Summers, Robert Rubin, Michael Blumenthal and George Shultz.
London mayor Boris Johnson, a leading campaigner for a so-called Brexit, has accused Obama of "outrageous and exorbitant hypocrisy", saying Americans would never accept the kind of control Brussels exerts over the EU.
A spokesman for the Vote Leave campaign told the BBC regarding the Treasury secretaries' letter: "Not content with doing down Britain's economy, No 10 (Cameron's office) are now soliciting help from across the pond."
Leaving the EU would also "likely disrupt and reduce trade flows" and while Britain would "no doubt" be able to forge new trade deals, this would not be easy, they said.
They repeated warnings by the IMF that uncertainty over Brexit was already affecting the economy in Britain, "and a vote to leave could introduce an extended period of uncertainty that could hinder growth even further".
They added that the world was facing major transnational issues such as financial crises, nuclear proliferation, pandemics and climate change that "no country, however powerful, can effectively address alone".
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