Open Arms, whose ship returned to the Cypriot port of Larnaca on Wednesday, condemned the deaths of the World Central Kitchen workers as "an incomprehensible act of violence".
The workers were moving food and supplies sent in a second shipload from Cyprus when their convoy was attacked on Monday.
"With the arrival yesterday of the Open Arms boat in Larnaca, Cyprus, the mission in the humanitarian corridor to the Gaza Strip with World Central Kitchen is suspended following the devastating attack suffered by the convoy," Open Arms said in a statement.
The NGO, which said 200 tonnes of food and supplies were delivered in the first shipment in March, expressed sorrow over the deaths.
"We demand answers and accountability for this unacceptable attack," Open Arms director Oscar Camps said in the statement.
World Central Kitchen, founded by Spanish-American celebrity chef Jose Andres, has suspended its work in Gaza because of the attacks. These have increased pressure on Israel over its treatement of Gaza's civilian population.
Israel has expressed regret over the attack and promised a transparent inquiry.
But major international aid groups have said it is now nearly impossible to work in Gaza, where Israel launched its military operation in response to the Hamas attacks on October 7.
The attacks resulted in the deaths of about 1,170 Israelis and foreigners, most of them civilians, according to an AFP tally of official Israeli figures.
Israel's retaliatory campaign to destroy Hamas has killed at least 33,037 people, mostly women and children, according to the Hamas-run health ministry in the Palestinian territory.
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