Ayatollah Ali Khamenei said in a speech posted online that the US was proposing talks while “pointing a gun at Iran”.
On Saturday, US Vice-President Joe Biden suggested direct talks; separate to the wider international discussions due to take place later this month.
But the US widened sanctions on Iran on Wednesday, aiming to tighten a squeeze on Tehran’s ability to spend oil cash.
Iran, which is subject to an array of international sanctions, has long argued that its nuclear programme is for energy generation and research.
Tehran’s critics believe the government is developing nuclear weapons.
The five permanent members of the UN Security Council plus Germany have held a series of negotiations with Iran over the years.
But they have often ended with Iran demanding the West lifts sanctions as a condition to any nuclear move, and the Western powers refusing.
Iran is expected to attend another round of negotiations on 26 February in Kazakhstan.
Mr Biden made his offer of direct talks during a security conference in Germany last weekend.
He said Washington was prepared to hold one-to-one talks with Iran “when the Iranian leadership, supreme leader, is serious”.
“That offer stands, but it must be real and tangible and there has to be an agenda that they are prepared to speak to. We are not just prepared to do it for the exercise,” he said.
On Sunday, Iranian Foreign Minister Ali Akhbar Salehi welcomed the offer, saying Iran had no “red lines”.
“But we have to make sure… that the other side comes with authentic intentions with a fair and real intention to resolve the issue,” he said.
The minister said Iran would take the offer into “serious consideration”, but the US had to desist from the “threatening rhetoric that everything is on the table”.
Iran’s supreme leader, however, said negotiations with the US “would solve nothing”.
“You are pointing a gun at Iran saying you want to talk. The Iranian nation will not be frightened by the threats,” he said.
President Mahmoud Ahmadinejad has several times announced he was ready for direct talks with the US.
But Ayatollah Khamenei’s advisers have criticised the president for making such a “submissive” offer, says the BBC’s Bozorgmehr Sharafedin.
The UN’s nuclear watchdog, the IAEA, has repeatedly accused Iran of not co-operating fully.
In its last report in November 2012, the agency expressed particular concern about activities at a facility in Parchin, just 20km (13 miles) from Tehran.
The agency wanted to investigate claims that Iran had built an explosive-containment vessel to conduct experiments there, saying such experiments “would be strong indicators of possible nuclear weapon development”.
But Iran refused to give inspectors permission to visit the site.
Iran’s repeated failure to ease international concerns has led to a gradual tightening of economic and political sanctions.
Analysts say Iran’s economy is being hobbled. Its currency, the rial, has seen its value plunge, and vital medicines are said to be in short supply.
Reprinted from BBC News