One of SeaWorld's most prominent orcas, Tilikum had been fighting a bacterial lung infection since March when he died at the marine theme park in Orlando, Florida.
SeaWorld, which owns several entertainment parks in the United States, said the approximately 36-year-old killer whale had spent years with the company.
"Tilikum passed away early this morning, January 6, surrounded by the trainers, care staff and veterinarians that provided him around-the-clock world-class care," SeaWorld announced on its website.
"Tilikum had, and will continue to have, a special place in the hearts of the SeaWorld family, as well as the millions of people all over the world that he inspired," SeaWorld president and chief executive Joel Manby said.
The official cause of death will not be announced until the completion of a necropsy, the company said.
It noted that Tilikum was near the upper end of the average life expectancy for a killer whale, and faced "some very serious health issues," it said.
The animal rights group People for the Ethical Treatment of Animals (PETA) tweeted a photo of Tilikum on Friday along with the message: "R.I.P. Tilikum Dead after three decades of misery."
Tilikum's name means "friend" in the Chinook Jargon language spoken among Native Americans in the Pacific Northwest.
The black-and-white five-tonne bull orca, the largest killer whale in captivity, had been linked to the deaths of three people.
In 2010, Tilikum grabbed the ponytail of Dawn Brancheau, a SeaWorld trainer, and dragged her underwater to her death in front of horrified onlookers at the end of a show at SeaWorld Orlando.
The whale, nicknamed "Tilly," was involved in the death of a part-time trainer at the Sealand of the Pacific facility in Canada in 1991 and that of a man who died in 1999 after he sneaked into SeaWorld to swim with the mammals after the park closed.
CNN featured Tilikum in a 2013 documentary "Blackfish" that covered those fatalities and also sought to show the impact of captivity on these giant sea creatures.
Animal rights groups charge that orcas were kept in tanks that are too small, fed improper diets and forced to perform tricks.
Under public pressure, SeaWorld last March announced it would stop breeding killer whales, and would no longer keep any of them in captivity after its current generation dies.
With the death of Tilikum, SeaWorld now has a total of 22 orcas at its three facilities -- in Orlando; San Antonio, Texas; and San Diego, California.