Israeli Prime Minister Benjamin Netanyahu aims to begin a process of annexing West Bank settlements and the Jordan Valley on July 1, as part of a US peace initiative.
"We call for the annexation project to be confronted with resistance in all forms," said senior Hamas official Salah Al Bardawil.
"We call on our people to transform this hardship into an opportunity to get the Palestinian project back on track," he told a press conference.
Deep divisions remain between Islamist movement Hamas and the Palestinian Authority, based in the West Bank city of Ramallah, but Bardawil called for a "union of the political class".
"It is the duty of each free Palestinian citizen to rise up against this flagrant aggression on our land," he said.
Bardawil called for a meeting between Hamas and the Palestine Liberation Organisation, which includes various other Palestinian groups.
Hamas took control of Gaza in 2007, after ousting forces of Palestinian President Mahmoud Abbas in a near civil war.
Both the PA and Hamas stand opposed to annexation in the West Bank, which forms part of a peace plan unveiled in January by US President Donald Trump.
The initiative paves the way for the eventual creation of Palestinian state, but on reduced territory and without key Palestinian demands such as a capital in East Jerusalem.
Israel’s intention to press ahead with annexation has been met with warnings from the United Nations that such a move would likely spark violence.