U.S. Agency for International Development subcontractor Alan Gross, 65, was convicted for illegally attempting to establish Internet service on the island and ended an 8-day hunger strike in April at the urging of his mother.
Evelyn Gross, 92, died in Plano, Texas, after a long battle with lung cancer, a statement from his lawyers said.
Her death is the latest blow to Gross and is likely to renew supporters' calls for his release.
"Evelyn Gross’s last wish was to see her son before she died. Cuban officials refused to give Alan a humanitarian furlough to visit his mother, despite repeated pleas and the certainty that she was dying," the statement said.
Alan Gross was arrested in 2009 while trying to establish an online network for Jews in Havana as a USAID subcontractor. It was his fifth trip to Cuba.
In 2011, a Cuban court sentenced him to 15 years in prison for illegally providing Internet equipment and service to Cuban Jewish groups under a U.S. program promoting political change that the Cuban government considers subversive.
Cuba has blamed the United States for Gross's incarceration and repeatedly offered to enter into talks without preconditions.
Previously, Cuba sought to link talks on Gross to the cases of three Cuban agents serving long prison terms in the United States for spying on Cuban exile groups in Florida. But the United States has rejected any trade of the Cuban agents for Gross, and no formal talks have taken place.
"This is a devastating blow for Alan and our family," Gross's wife, Judy Gross, said in the statement. "I am extremely worried that now Alan will give up all hope of ever coming home and do something drastic. Surely, there must be something President Obama can do to secure Alan's immediate release."
(Reporting by Daniel Trotta; Editing by David Adams and Susan Heavey)