The negotiators, who resumed talks with world powers in Vienna on Tuesday, "are working, making efforts, breaking sweat... to obtain what is in the interests of the country and the regime", he said in a meeting with members of parliament.
The intervention from Khamenei, who has the final say on all matters of state in Iran, came after several deputies accused the negotiators of having betrayed the Islamic republic by making key concessions.
In footage filmed on a mobile phone and widely circulated on social media, ultra-conservative MP Mehdi Kouchakzadeh went so far as to accuse Foreign Minister Mohammad Javad Zarif of "treason" as leader of the negotiating team.
But Khamenei, quoted on his website, said: "On nuclear issues, our positions are clear and the same as I have expressed publicly... These are the positions of the regime."
Critics accuse negotiators of having accepted demands for international inspections of Iran's nuclear sites, a position which Khamenei has totally ruled out.
Zarif has said "some access" could be allowed under an Additional Protocol of the Nuclear Non-Proliferation Treaty (NPT), but not inspections of military sites.
In his bid to restore order on the home front, Khamenei called for "mutual respect" -- without accusations and suspicions -- between parliament and the government.
Members of parliament must maintain a "respectful attitude towards ministers" and the government must avoid insulting deputies, he said.
Iran and the P5+1 group -- Britain, China, France, Russia, the United States plus Germany -- signed a framework agreement on Tehran's controversial nuclear programme on April 2.
They aim to seal a final deal by the end of June to prevent Iran from developing an atomic bomb, in exchange for easing crippling economic sanctions.
Gulf in the Media