Khamenei's latest salvo comes as US Secretary of State Hillary Clinton is set to meet Palestinian leaders on Wednesday during her first visit to the region since taking office.
She was to hold meetings with Western-backed president Mahmud Abbas and prime minister Salam Fayyad in the occupied West Bank's political capital of Ramallah a day after huddling with Israeli leaders in Jerusalem.
"The only way to save Palestine is resistance," Khamenei said in his address to a gathering of officials from the international community in Tehran.
"Support and help to Palestinians is a mandatory duty of all Muslims. I now tell all Muslim brothers and sisters to join forces and break the immunity of the Zionist criminals."
He also called for putting Israeli leaders on trial for the recent assault on Gaza.
"The political and military leaders of the occupying regime that played the role in the disaster of Gaza must be put on trial. This is the first step that should be taken," Khamenei said.
Iran does not recognise Israel and Khamenei has repeatedly refused a two-state solution to solve the Israel-Palestinian issue.
Khamenei said any negotiations to solve the issue was fruitless, adding the United States and Britain committed the "crime of creation and supporting this cancerous tumour (Israel)."
"Even the new president of the United States who came to power with the motto of changing the (George W.) Bush administration's policies talks about unconditional commitment to secure Israel. This is defending terrorism by a government."
Obama has vowed to actively pursue the peace process and has appointed a special envoy, veteran diplomat George Mitchell, to coax both sides back to the negotiating table.
The Tehran summit comes just two days after a global donors meeting in Egypt pledged to donate around 4.5 billion dollars to rebuild Gaza.
"The (Tehran) conference is to discuss the issue of aiding Palestinians and also find a solution to the Zionist regime's adventures against Palestinians," Ali Larijani, speaker of Iran's parliament, told FARS news agency.
Iran is an arch-foe of Israel and does not recognise its existence. It is a close ally of the Islamist Hamas movement that controls Gaza.
Tehran has also strongly criticised Israel's deadly 22-day offensive in Gaza in December-January that killed 1,300 Palestinians and also 13 Israelis.
It also criticised the Egypt conference.
"Those who participated in that conference and staged that puppet show approved the Zionist regime's atrocities in Gaza," Ali Akbar Mohtashamipour, the secretary general of the Iran conference said, according to Fars agency.
The summit in Tehran also comes three days after Iran's chief prosecutor Ghorbanali Dorri-Najafbadi called for arrest warrants to be issued against 34 Israeli political leaders and 114 military commanders.
He mainly targeted outgoing Israeli Prime Minister Ehud Olmert, Defence Minister Ehud Barak and Foreign Minister Tzipi Livni for committing "war crimes."
