Laziz (Cutey in Arabic) the tiger and the 15 other remaining animals from the Khan Yunis zoo were driven on a trailer in cages to the Erez crossing before dawn, an AFP journalist said.
The animals crossed the border en route to sanctuaries and zoos in South Africa, Jordan and Israel for "a better quality of life", the Israeli army said in a statement.
As well as Laziz, five monkeys, two emus, two deer, two eagles, two turtles, a pelican and a porcupine were transferred, the Four Paws charity ensuring the animals' welfare said.
Dozens of animals had died in the zoo, some of starvation, and badly stuffed bodies of dead crocodiles, lions and others were left in the open, surrounded by piles of bones.
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The NGO says on its website that Khan Yunis had "been known as the 'worst zoo in the world' since it became public last year that the zoo was crudely mummifying the animals that died in their care and displaying them."
"So many people have followed the progress of our mission eagerly and shown their solidarity with Laziz and the other animals," Four Paws vet Amir Khalil said in a statement.
"We are happy that we were finally able to close down Khan Yunis zoo."
Laziz's case attracted particular attention, after it was revealed the near-starving tiger had originally been brought to Gaza from Senegal via Egypt, where he was smuggled through tunnels into the enclave.
He will now be transferred to the Lionsrock refuge for big cats in South Africa.
"But the tiger just went straight into the box as if he knew that we will bring him to a nice home."
In the years after its 2007 opening there were more than 100 animals housed at the zoo, but they were decimated by repeated wars and shortages of customers.
The owners were unable to afford enough food, with some animals starving.
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