The protesters demanded Prime Minister Ariel Sharon put the pullout plan to a referendum.
Settler leaders said Sunday's demonstration was the biggest since Sharon announced his plan last year.
Sharon's initiative to remove all the Jewish settlements from Gaza and four of the 120 in the West Bank has helped revive hopes for a new Middle East peace.
But the plan to give up some of the land seized in the 1967 war has roused fears of violent confrontations between the Israeli government and Jewish settlers.
Protesters massed near the Israeli parliament as a helicopter hovered, and security was tightened around the prime minister's office nearby.
But chances for preventing a pullout have decreased with Sharon's formation of a unity government late last year with the left-wing Labour Party and with several opposition parties vowing to support the plan from outside his cabinet.
"It is clear to me this will not pass quietly. I feel that people will be hurt during the evacuation"
Tel Aviv resident Shalom Fleiser
"I don't know if this will help but at least we can express how we feel and we hope we will not be ignored," said Galit Kahune, a Gaza settler. "I very much fear this [withdrawal] will create a rift among the people."
Hardliners and settler leaders have called for a nationwide referendum on the plan.
Sharon has ruled out such a move, saying opponents are just trying to delay the pullout, set to start this year. But some Likud members - including his main rival and Finance Minister
Benjamin Netanyahu - are in favour of a referendum.
Hardliners have warned of clashes between settlers and Israeli security forces during a pullout, and have urged soldiers and police officers to disobey orders to take part.
Brink of war
"It is clear to me this will not pass quietly," said Tel Aviv resident Shalom Fleiser. "I feel that people will be hurt during the evacuation.
Some settlers fear the pullout will
cause a rift among Israelis
"This situation will lead us to the brink of civil war," said Yair Kali, 48, of the West Bank settlement of Psagot.
The largest anti-pullout protest was in July when more than 100,000 Israelis joined hands in a 90km human chain from Jerusalem to the Gaza Strip.
About 8000 Jewish settlers live in heavily protected colonies in Gaza alongside more than 1.3 million Palestinians. More than 230,000 settlers live in the West Bank, home to 10 times as many Palestinians.
Palestinians would welcome an Israeli withdrawal from any of the territory they want for a state, but fear Sharon's plan is a ruse to keep hold of the much larger West Bank settlements.
New Palestinian President Mahmud Abbas has said he would coordinate with Israel on the pullout if it is part of broader talks on statehood.