The date 17 March is also the day the Knesset is to deliberate on the pullout plan introduced by Israeli Prime Minister Ariel Sharon.
Emily Amrusy of the Yesha Settler Council said on Sunday the day was chosen according to the Hebrew calendar because it marks the eve of the death of the prophet Moses, who led the Israelites to the holy land.
Many settlers claim Gaza as part of a biblical heritage.
"For the first time in the country's history, great rabbis of religious Zionism have called a day for fast and prayers, by the authority of Jewish law," Amrusy said in a statement.
She said the fast was a protest against the planned removal of more than 8000 Jewish settlers from 21 illegal Jewish settlements in the Gaza Strip and four in the northern West Bank this summer.
She said it aimed to "destroy the expulsion order."
Sharon is also expected to bring his budget to the Knesset for final approval on 17 March in a vote which could determine whether the pullout happens.
Far-right rebels in Sharon's Likud party who oppose removing the settlers have vowed to vote against the budget. If he fails to get the budget passed by 31 March, new elections would have to be called and the pullout likely put on hold.
Israel's Knesset gave approval for the Gaza withdrawal last month by ratifying a plan to compensate settlers who would be removed.
The withdrawal is set to begin on 20 July and last four weeks.