In a 10-minute speech uploaded to jihadist forums, Mr Zawahiri argued that the Pakistani authorities only represented US interests, according to a statement from SITE Intelligence Group.
Zawahiri, shown standing in front of a green curtain, urged Pakistanis to follow the example of the Arab Spring as the military could not be expected to turn against America despite a deadly US strike on Pakistani troops in November.
"O our brothers in Pakistan! O our people in Pakistan! This treacherous army and bribe-taking government have plundered your wealth," he said in the video.
"They have ruined your economy and destroyed your world as well as your hereafter. What then are you waiting for?
"Take a lead from your brothers in Tunisia, Egypt, Libya, Yemen and Syria who are standing up against oppression and oppressors with their bare chests exposed and offering sacrifices so that victory may be ordained for then."
Mr Zawahiri also said that unless its demands were met, Al-Qaeda would not release an elderly American development expert who was captured in Pakistan last August.
"By the grace of Allah, we, on our part, have captured the American Jew Warren Weinstein.
"He will not return to his family, by the will of Allah, until our demands are met, which include the release of Aafia Siddiqui, Sheikh Omar Abdul Rahman, the family of Shaikh Osama bin Laden, and every single person arrested on allegations of links with Al-Qaeda and Taliban."
Long Al-Qaeda's number two, Mr Zawahiri took over the helm of the group after Bin Laden was killed in May 2011 in a US special forces night raid deep in Pakistan.
The video is the latest of a number in which the militant chief has attempted to exploit the Arab uprisings.
Al-Qaeda has been absent from the popular protests that swept the Arab world last year, leading to the ouster of autocratic, secular leaders in Egypt, Libya and Tunisia, and sparking unrest elsewhere.
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