"Salvation of the Muslim nation is through the march of its sons on the path of jihad," he says in the clip posted online on Wednesday.
A banner accompanying the 11-minute audio file says it marks the 41st anniversary of the 1967 Middle East war which Arabs refer to as the "naksa" or "setback".
Israel occupied the West Bank, including Arab East Jerusalem, Gaza, the Sinai and the Golan Heights in the war.
Al-Qaeda has vowed attacks on Jews both within and outside Israel.
The audio clip allegedly by al-Zawahiri follows recent statements attributed to Osama bin Laden, al-Qaeda's leader, placing more emphasis on the Palestinian issue.
Last week, bin Laden urged Muslims to break the Israeli-led blockade of the Hamas-controlled Gaza Strip and fight Arab governments that deal with Israel.
In the last message attributed to him on April 22, 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.