Around 1,000 settlers massed inside the main entrance to Neve Dekalim in the first sign of active resistance since Israel’s official launch of its pullout from the territory.
The settlers were singing, praying and calling on soldiers and police to disobey their orders.
“Soldier, policeman, refuse the orders,” they chanted in unison as troops looked on.
The settlers are being given 48 hours to leave or face forcible expulsion.
While military sources said notices have already been handed in some of the smaller settlements such as Nissanit and Elei Sinai in northern Gaza, troops have not even started attempting to deliver notices in Neve Dekalim, according to AFP.
Israel’s operation to leave Gaza after a 38-year occupation formally began at midnight when the army outlawed the continued presence of any settlers in the 21 settlements.
Meanwhile, Israel said it intends to retain control of six West Bank settlement blocs in a final peace deal with the Palestinians.
“The settlement blocs in Judea and Samaria (the West Bank) and the Jordan valley will remain under Israeli control,” said Defence Minister Shaul Mofaz, speaking on army radio.
Mr Mofaz also singled out the communities of Maale Adumim and Gush Etzion, both of which are close to Jerusalem, as well as three areas in the northern West Bank and the settlement of Efrat near Bethlehem.
He said while the operation to uproot the Gaza settlers is painful, it is vital that laws must be respected.
“The state has decided and now it is the time to carry out that decision,” said Mr Mofaz.
“This is breaking our hearts but it is vital for the future of the democratic state of Israel.”