"The UK is an extraordinary partner for America and a force for good in an unstable world. I hope it remains strong, robust and united," Obama tweeted Wednesday night.
The last-minute intervention by the US president is seen as an indication that Washington is concerned one of its closest allies, the United Kingdom, could break up.
More than 4.2 million registered voters are casting their ballots in Scotland’s landmark referendum on Thursday.
Voters will decide on ending their more than 300 years of union with the rest of the UK, made up of England, Wales and Northern Ireland.
Obama first announced his stance on the vote in June.
"We obviously have a deep interest in making sure that one of the closest allies that we will ever have remains strong, robust, united, and an effective partner," Obama told reporters after the G7 summit. "But ultimately these are decisions that are to be made by the folks there."
Other officials in Washington have also urged Scots to vote no, saying independence would diminish the United Kingdom’s power on the world stage.