"Do not give up... increase your martyrdom attacks, the number of rockets you launch and your ambushes. There is no other solution," Zawahiri said in the message marking the anniversary of the June 1967 Arab-Israeli war.
Gaza has been under a punishing Israeli blockade since the Islamist movement Hamas seized power in the impoverished Palestinian territory last June, in a move Israel says is aimed at halting rocket attacks.
"To those who try to make you despair, tell them... America is being defeated by our brothers in Iraq and Afghanistan. Why should we despair," said Zawahiri, who has a 25 million dollar US bounty on his head.
"This naqsa (setback) has shown that the salvation of this Muslim nation would come through the jihad (holy war) of its people... The governments have already betrayed" their people, he said.
The term "naqsa" is used by Arabs to refer to the defeat of the armies of Egypt, Jordan and Syria in 1967, which led to Israel's occupation of the West Bank and Gaza, Syria's Golan Heights and Egypt's Sinai, which was returned to Egypt after signing the Camp David peace accords in 1978.