Gaza has been sealed off from the outside world since Israel began its air and ground invasion five months ago and the United Nations has warned of widespread starvation.
The charity ship Open Arms sailed out of the port of Larnaca towing a barge containing flour, rice and protein.
The 200-mile journey to Gaza could take as long as two days because of the heavy tow barge.
Bureaucratic obstacles and insecurity have hampered aid deliveries since the start of the war on October 7.
Aid agencies say the latest resort to sea routes and airdrops can provide only limited relief since Israel has sealed off most land crossings.
That was also the mood among skeptical Gazans on the beach at Rafah, near the southern border with Egypt.
Hussein Atallah, displaced like most people in the enclave, said aid should be able to cross by land.
"Also the border between us and Egypt is 15 kilometers long. So why the port then? This is the question, why would they do a port here?"
The aid ship was funded mostly by the UAE and organized by U.S.-based charity World Central Kitchen.
With much of Gaza reduced to rubble, WCK said it was building a landing jetty with material from destroyed buildings.
Open Arms spokesperson Laura Lanuza:
"For us is very important, not only because this is the first boat that is going to arrive in Gaza via these maritime corridor, because we know there has been a naval blockade from Israel since over 20 years ago. So for us, it makes us really, really happy and proud of that this is happening."
Qatar's foreign ministry spokesperson Majed Al-Ansari said on Tuesday negotiators seeking a ceasefire between Israel and Hamas were not close to a deal.
There'd been hopes for a truce in time for Ramadan, which began this week.
More than 31,000 Palestinians have been killed since Israel launched its war on October 7, following an attack by Hamas militants that killed 1,200 people in Israel and took 253 hostages, according to Israeli tallies.
