Sunday's protest - the biggest since Prime Minister Ariel Sharon unveiled his plan in February - highlighted the stiff domestic opposition to the withdrawal initiative.
"This chain is a sign of strength," said Anita Tucker, one of the 8000 settlers living in heavily protected Gaza enclaves alongside more than 1.3 million Palestinians.
"Most of the people love the land of Israel and don't want to see anything destroyed," she said.
Organisers said some 200,000 people took part, while police estimated the crowd at 130,000.
Under Sharon's disengagement plan, all 21 settlements in Gaza and only four of the 120 settlements in the occupied West Bank would be evacuated by the end of next year.
The 90km human chain stretched from the northern Gaza Strip to Jerusalem's Western Wall. Police ordered an eight kilometre gap outside Jerusalem for security reasons.
Bus-loads of protesters descended on Jerusalem, many waving Israeli flags.
"We are expressing the fact that all of Israel belongs to all the people of Israel," said a protester.