The Shireen vessel, a legal support boat part of the Gaza-bound Global Sumud Flotilla departed at 9:30am on Sunday morning, from the Kato Paphos harbour where it was docked.
According to a port authority official, the delay in the departure was due to a crew-change process.
A Canadian mechanic who was on board did some repairwork on the vessel. Other passengers expressed a wish to leave Cyprus via flight, requiring a crew change.
After the vessel was refuelled and stocked up with water, it departed at 9:30am to Italy.
The Shireen had docked on Friday night in Cyprus, after being the Sumud Flotilla was intercepted by Israeli forces.
On Thursday at around 11pm a vessel that was also participating in the flotilla transporting humanitarian aid to the Gaza Strip submitted a request and eventually docked at the port of Larnaca.