Zawahiri made the appeal in an audio message entitled "Who Will Avenge the Scientist Aafia Siddiqui," which was released on militant forums, the US-based SITE Intelligence Group said.
He promised "to attack ... (Americans) as long as they attack" Muslims and said "the ummah will not stop pursuing you."
Zawahiri also told Pakistanis their "government humiliated them by letting the Americans and Crusaders occupy the country."
He called for them to "take the only available path, that of jihad ... which will liberate Aafia Siddiqui."
On September 23, a New York court jailed Siddiqui for the attempted murder of US servicemen in Afghanistan, in a high-profile case sparking outrage in Pakistan.
Siddiqui, a mother of three, was found guilty of grabbing a rifle at a police station in the Afghan town of Ghazni where she was being interrogated in July 2008 and of trying to gun down a group of US servicemen and FBI agents.
Prosecutors said she opened fire, shouting "death to America!"
She did not hit anyone and was herself shot in the stomach before being subdued.
Siddiqui, a neuroscientist who trained at the prestigious Massachusetts Institute of Technology and Brandeis University, repeatedly pleaded in court for Muslims to stay calm.
"Forgive everybody in my case, please ... And also forgive Judge Berman," she said.
Zawahiri's last message was in July, when he denounced France's move to ban women from wearing the Islamic face veil in public.
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