The ship, operated by the Spanish aid group Open Arms, left Cyprus on Tuesday towing a barge laden with food sent by World Central Kitchen, the charity founded by celebrity chef José Andrés.
It could be seen off Gaza's coast Friday morning.
The United States has joined other countries in airdropping supplies to the isolated region of northern Gaza and has announced separate plans to construct a pier to get aid in.
Aid groups said the airdrops and sea shipments are far less efficient ways of delivering the massive amounts of aid needed in Gaza.
Instead, the groups have called on Israel to guarantee safe corridors for truck convoys after land deliveries became nearly impossible because of military restrictions, ongoing hostilities and the breakdown of order after the Gaza police force largely vanished from the streets.
The daily number of supply trucks entering Gaza since the war began has been far below the 500 that entered before 7 October.
Earlier in the week, Israel allowed only six aid trucks to enter directly into the north, a step aid groups have long called for.
World Central Kitchen operates 65 kitchens across Gaza from where it has served 32 million meals since the war started, the group said.
It plans to distribute the food in the north, the largely devastated target of Israel’s initial offensive in Gaza, which has been mostly cut off by Israeli forces since October.
Up to 300,000 Palestinians are believed to have remained there despite Israeli evacuation orders, with many reduced to eating animal feed in recent weeks.
The aid is a tiny fraction of what is required, but the shipment was intended to pave the way for other larger shipments, officials working on the route have said.
A second vessel being loaded with even more aid will head to Gaza once the aid on the first ship is offloaded and distributed, Cyprus’ Foreign Minister Constantinos Kombos said.
He declined to specify when the second vessel would leave, saying it depends in part on whether the Open Arms delivery goes smoothly.
Israel’s war in Gaza has killed over 31,000 Palestinians and driven most of Gaza’s 2.4 million people from their homes. A quarter of Gaza’s population is starving, according to the United Nations.
The ship could be spotted from the coast hours after the Israeli forces launched a new attack near an aid distribution point in northern Gaza, killing 20 people and wounding 155 others.
The health ministry said a group waiting for aid near the Kuwaiti roundabout was hit by Israeli shelling late Thursday.
Bloodshed surrounding an aid convoy on 29 February killed 118 Palestinians in northern Gaza.
Witnesses and hospital officials said most of the casualties were from bullet wounds.
After that, plans for the sea route took shape, and other countries joined Jordan in dropping aid into the north by plane.
But people in northern Gaza say the airdrops are insufficient to meet the vast need. Many can’t access the aid because people are fighting over it, said Suwar Baroud, 24, who was displaced by the fighting and is now in Gaza City. Some people hoard it and sell it in the market, she said.
A recent airdrop that malfunctioned plummeted from the sky and killed five people.
Another drop landed in a sewage and garbage dump, said Riham Abu al-Bid, 27. Men ran in but were unable to retrieve anything, she said.
“I wish these airdrops never happened and that our dignity and freedom would be taken into consideration, so we can get our sustenance in a dignified way and not in a manner that is so humiliating,” she said.
The war has exacerbated tension throughout the region and threatened to flare into broader violence.
At Al-Aqsa Mosque in occupied Jerusalem, the first Friday prayers of the Muslim holy month of Ramadan were being held amid Israeli restrictions on worshipers.
The mosque has been a frequent flashpoint for Israeli-Palestinian violence in the past, with Israeli police and settlers storming the coumpound.
Israel put restrictions in place limiting West Bank Palestinians’ access to the compound for Friday’s prayers to men over 55, women over 50 and children under 10.
Short link: