The stakes are so high that the government has reportedly released six members of parliament, currently serving jail terms, to vote.
Singh has expressed confidence that his government will survive.
He has assured members of parliament that the nuclear agreement was done "in the best interests of our people".
The government will need a simple majority of votes, but opposition parties, including the Bharatiya Janata Party (BJP), a conservative party, are equally confident.
"The government is today like a patient in an intensive care unit. The first question naturally asked is, 'is he going to survive or not?'" Lal Krishna Advani, the BJP leader, said.
India tested nuclear weapons in 1974 and 1998 and refuses to sign the nuclear Non-Proliferation Treaty, and is currently barred from buying nuclear technology and fuel.
The deal would allow such purchases but subject India's civilian nuclear sites to international controls such as UN inspections, said to ensure that any purchases are not diverted for military use.
Opponents say the agreement will compromise India's position as a politically neutral country, and that the requisite UN inspections would limit the country's ability to develop its weapons programme and deter Pakistan, its main regional rival.
They also argue that there are strings attached, and doing a deal with the US would undermine its freedom to buy oil and gas from countries such as Iran, or shop for armaments with traditional suppliers including Russia.
Todd Baer, Al Jazeera's correspondent in Delhi, said: "This is the first time in India's history where a major foreign policy issue will decide the country's political future.
"Should the government of Manmohan Singh fall fresh elections will be called ... a good six months before national polls were due."
The nuclear deal is a topic spanning India's energy security as well as its place in the world.
"We are not against nuclear energy. We are not against a very close relationship with America. But we would never like India to become party to an agreement which is unequal," Advani said during Monday's debate in parliament.
"This deal makes us a subservient partner. It makes India a junior partner."
The BJP and the communists are also trying to widen the terms of the debate.
They have been speaking out against rising food and fuel prices - inflation is currently around 12 per cent - and arguing that hundreds of millions of the poorest people have been left behind in India's economic boom.
Speaking to Al Jazeera, Pramit Pal Chaudhuri, of The Hindustan Times daily, said that regardless of whether or not it survives the confidence vote, the government must hold elections within six months in accordance with the Indian constitution.
He said that most of the Indian electorate is "unconcerned" with the nuclear deal, but instead are worried about the high level of inflation.
Chaudhuri predicted that the Singh government will narrowly survive the vote, but said that in any case, the electorate will remain "angry" owing to inflation.