Hamas spokesman Mushir al-Masri said on Friday that an attack on the Kfar Darom colony came in response to Israeli aggression.
"Amid continued Zionist crimes, we find ourselves with no choice but to defend our people by all means available," he said.
An Israeli military spokesman confirmed three Palestinian fighters took over a house near the heavily fortified colony and opened fire with light arms and anti-tank weapons.
One of the three was killed and another was wounded in the ensuing gun battle with Israeli troops, Palestinian sources said.
A third attacker was believed to have fled. There were no reports of Israeli casualties.
The clash came one day after Israel's defence chief Shaul Mofaz ordered the army to use all necessary means to stop home-made mortar and rocket launches on illegal settlements.
And an official in Prime Minister Ariel Sharon's office, David Baker, added: "The Palestinian Authority allows these terrorists to roam freely and strike at Israeli civilians within their communities ... Israel is ready to take appropriate steps to defend its citizens."
Israeli troops killed a Hamas member earlier on Wednesday in a missile strike on a group firing mortars at nearby settlements. A second Palestinian was killed under disputed circumstances.
Since the signing of the Sharm al-Shaikh ceasefire agreement on 8 February, 23 Palestinians have been shot by Israeli soldiers - 11 of them children. No Israelis have been killed in the same period.
West Bank raid
Meanwhile, in the West Bank, Israeli occupation forces launched an operation in the village of Syda, north of Tulkarim, and arrested four Palestinians, Aljazeera's correspondent reported.
In the Balata refugee camp, east of Nablus, three Palestinians were wounded by Israeli gunfire and seven others arrested on Friday.
The clashes on Friday further strained a shaky three-month truce.