Syria's President Bashar al-Assad belongs to the Alawite sect of Shiite Islam, while the divided rebels fighting to oust his regime are mainly Sunni Muslims, including the jihadist Al-Nusra Front.
"Unite, listen and agree that you shall not lay down your arms and leave your trenches until the establishment in the Levant of an Islamic state that will restore the caliphate," said Zawahiri.
"The United States, it associates and allies want you to sacrifice your blood... To bring down the criminal Alawite regime and install a government in its pay and to preserve Israel's security," he said in the message posted on Islamist websites today.
"Your laudable jihad gives hope for the recovery of Palestine, 65 years after it was robbed from us," he added in the message marking the anniversary of the Six-Day War in which Israel seized the West Bank and Gaza Strip.
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Syria's civil war flared after Assad's forces launched a bloody crackdown on democracy protests that erupted in March 2011.
Tens of thousands of Syrians have since been killed and millions forced to flee their homes.
This assertion was bolstered in April when Al-Nusra Front leader Abu Mohammed al-Jawlani pledged allegiance to Zawahiri.
The vow came after the Al-Qaeda chief urged rebels to fight for the establishment of an Islamic state in Syria, a call that was met with caution by the mainstream rebel group, the Free Syrian Army.