"Nobody should dare to threaten humanity this way. We will not allow the US to impose its infernal domination on Iran again with these threats," Ayatollah Khamenei said, quoted by Fars news agency.
Khamenei, speaking to a group of nurses, targeted Obama and said the international community should react, referring to a new US nuclear policy which bars the use of nuclear arms but singles out Iran and North Korea as "exceptions."
Obama's "nuclear threat marked a black spot in that government's record," said Iran's leader, whose country has interpreted the US policy as tantamount to a threat of nuclear attack.
Khamenei lashed out at Washington, saying the arch-foe's attempt to initiate "friendship" with Tehran last year was nothing but an example of the "wolfish" and "bloodthirsty" nature of the United States.
Obama, in an unprecedented Persian New Year message to the Islamic republic in 2009, offered a hand of diplomacy to help ease the animosity between the two enemies.
"It is clear what is behind this … outreaching of a hand of friendship," Khamenei said.
"It is evident now what is at work behind the scene and what their bloodthirsty and domineering nature is. These threats will get nowhere. Iranians will get them down to their knees."
Tehran and Washington have had no diplomatic relations since the aftermath of the 1979 Islamic revolution.
The animosity has grown over Tehran's defiant pursuit of a controversial nuclear programme which Washington suspects masks a weapons drive, an allegation denied by Iran.
President Mahmoud Ahmadinejad last week called for Washington's suspension from the International Atomic Energy Agency along with all other nations which possess nuclear arms.
