Khamenei, in a televised address, also called any talks with the United States a "dead end" that were of no benefit to his country.
His statement came as European powers seeking to reimpose tough sanctions on Iran over its nuclear programme met with the Islamic republic's top diplomat, without any signs of a compromise.
"The American side has been adamant that Iran must not have (uranium) enrichment," Khamenei said.
"We did not surrender and we will not. We did not and will not yield to pressure in this matter or any other matter," he added.
The supreme leader went on to say talks with the United States would only bring harm to Iran.
Negotiating with America "not only brings no benefit, but also causes major harm under the current conditions, some of which might even be described as irreparable", he said.
"Let us not forget the experience of these past 10 years, and the other side -- which is the subject of our discussion for now -- is the United States," he continued, referring to the landmark 2015 nuclear deal that Washington withdrew from three years later.
"This other side consistently breaks promises, lies about everything, deceives and threatens militarily at every opportunity," Khamenei said.
"One cannot negotiate with such a party. In my view, negotiations with the United States on the nuclear issue, and perhaps on other issues as well, are a complete dead end."
European countries and the United States suspect the Iranian government of seeking to acquire an atomic bomb. Tehran has vigorously denied that, insisting it has a right to civilian nuclear energy.
Israel in June carried out a major military campaign against Iranian nuclear sites, with President Donald Trump joining by ordering US warplanes to drop bombs on key targets.
The Trump administration, which had long pushed for the reimposition of sanctions, has voiced a willingness to hold talks with Iran, which doubts Washington's sincerity.
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