Just one Pacific bluefin tuna is remaining in a tank at the Tokyo Sea Life Park, near Tokyo Bay.
The 2,200-ton tank, which was once home to nearly 160 bluefin tuna, eastern little tuna and oriental bonito, had been one of the park's biggest attractions.
But the fish began dying in November, and by January only 11 bluefin tuna remained alive.
The penultimate fish was found dead inside the tank last week, leaving one sole survivor.
It had suffered a broken backbone after hitting the aquarium wall twice.
A virus was discovered among some of the dead fish, but it is not thought to have been behind the mass deaths.
Other theories behind the mystery include lighting inside the tank and a possible poisonous substance in the water.
A spokesman for the park said last week: "We have had the tuna tank since the aquarium opened in 1989 but never experienced this kind of mass-dying.
"We are studying what caused the fish deaths, but we haven't figured it out yet.
"We suspect it could be due to new factors that were not present before."
Pacific bluefin tuna are an endangered species due to overfishing, and the aquarium had a breeding programme intended to help their numbers.