In the audio clip, the speaker told Palestinians: "Step up your martyrdom-seeking operations, and increase your missiles and ambushes, as there is no solution but this.
"Salvation of the Muslim nation is through the march of its sons on the path of jihad."
The speaker also branded Hosni Mubarak, Egypt's president, a traitor for not opening his borders to Palestinians.
"The brother from Gaza is refused entry [by Egypt], while an Israeli tourist is allowed to enter without a visa," he said.
Al-Qaeda has vowed attacks on Jews both from within, and outside, Israel.
The audio clip follows recent statements attributed to Osama bin Laden, al-Qaeda's leader, placing more emphasis on the Palestinian issue.
Last week, a tape reported to be from bin Laden tape urged Muslims to break the blockade of the Hamas-controlled Gaza Strip and fight Arab governments that deal with Israel.
Al-Zawahiri urged Muslims to support fighters from the Islamic State in Iraq group, which he said posed the biggest threat to US-led forces in the country, in the last message attributed to him on April 22.