The move comes amid tensions with Turkey over its activities in the area and a maritime deal with Libya expanding Ankara's claims over a large gas-rich area of the sea.
The 2,000-kilometre (1,200-mile) EastMed pipeline will be able to carry between nine and 12 billion cubic metres of gas a year from offshore reserves held by Israel and Cyprus to Greece, and then on to Italy and other southeastern European countries.
The discovery of hydrocarbon reserves in the eastern Mediterranean has sparked a scramble for the energy riches and a row between Cyprus and Turkey, which occupies the northern part of the Mediterranean island.
Greek Prime Minister Kyriakos Mitsotakis, Israeli Prime Minister Benjamin Netanyahu and Cypriot President Nicos Anastasiades joined the ceremony at which their respective energy ministers signed the deal in the Greek capital.
The EastMed project is expected to make the three countries key links in Europe's energy supply chain.
It could also help counter Turkey's effort to extend its control to the eastern Mediterranean.
Turkey already faces European Union sanctions over ships searching for oil and gas off Cyprus, whose government in Nicosia is not recognised by Ankara.
Turkish President Recep Tayyip Erdogan said in November he envisaged joint energy exploration activities with Libya in the eastern Mediterranean.
His government insists the Turkish Republic of Northern Cyprus -- recognised only by Ankara -- has the right to explore around the entire island.
Vice President Fuat Oktay said Turkey's pact with Libya had allowed Ankara to foil a plot to confine the country to land.
"No matter who is involved, no plan in the region which excludes Turkey has any chance of success," Oktay told the Anadolu state news agency.
Greece responded angrily to the Turkey-Libya deal, expelling the Libyan ambassador and urging the UN to condemn it.
Part of the agreement sets a maritime boundary between the two countries, which Greece says fails to take into account the Greek island of Crete.
'Peace and cooperation'
The EastMed alliance "is of enormous importance to the state of Israel's energy future and its development into an energy power and also from the point of view of stability in the region," Netanyahu said in a statement issued as he left Israel for Greece Thursday.
Mitsotakis said the pipeline was of "geo-strategic importance" and would contribute to regional peace.
Earlier, Greek Energy Minister Kostis Hatzidakis called it "a project of peace and cooperation" despite "Turkish threats".
Anastasiades said his aim was "cooperation and not rivalry in the Middle East".
Avinoam Idan, a former Israeli government security official who is now a geostrategy expert at Haifa University, said of the deal: "It's important for Israel, it's important for the transit countries, Greece and Cyprus, and of course Europe".
As the new source of energy would not compete with Russian supplies to the EU, "there is no reason to see it as a big change in the geopolitical dynamic in Europe's energy market", he told AFP.
The Greek economic daily Kathimerini said Wednesday that Athens and Nicosia had been in a hurry to finalise EastMed so as "to counter any attempt by the Turkish neighbour to stop the project".
The cost of the installation from the eastern Mediterranean to Italy is estimated at 6.0 billion euros ($6.7 billion).