The tiger, who was 14 years old, and had been at the resort attraction for eight years, will be hugely missed his handlers said tonight.
Zambar died earlier today. The zoo revealed he had been suffering from health problems for six months.
Darren Webster, director at Blackpool Zoo, said: "It is with great sadness that we can confirm that Zambar, our beloved male Amur tiger, has passed away.
"Zambar has been receiving specialist treatment from keepers and vets for various health problems for six months.
"His condition worsened this week and he died peacefully in his enclosure earlier today.
"He arrived in Blackpool in 2010 after being born and hand reared in Marwell Zoo in 2004. He was joined by Alyona in 2012 and they became parents to Barney and Radzi in 2014. Alyona is being closely monitored by her keepers.
"He was a hugely popular member of our zoo family and was loved by all of our visitors. Staff and keepers are extremely saddened at his passing and are being comforted by the kind messages of condolence we are receiving."
The pair's cubs have grown and moved on to other zoos in Europe, which leaves Alyona on her own.
A social media message from the zoo attracted hundreds of replies from well-wishers.
Lydie Taylor posted: "We are so sad this evening reading this news. Zambar will always have a special place in our hearts and we will miss him on our visits to the zoo. We as members enjoyed making enrichment boxes for him and I have a cast of his footprint too. He was such a lovely lad, we convinced ourselves he knew us when we called his name. We were so excited when he came to the zoo, and have followed his journey since. Sending our thoughts at this sad time to you all and the Zambar loving community."