Sudan, 45, lived at the Ol Pejeta Conservancy in Kenya. He was put to sleep on Monday after age-related complications worsened significantly.
His death leaves only two females – his daughter and granddaughter – of the subspecies alive in the world.
Hope for preserving the northern white rhino now lies in developing in vitro fertilisation (IVF) techniques.
Attempts to use Sudan to prolong the species with the two surviving female rhinos had failed, and an account was created for him on the dating app Tinder last year, not to find love, but to help fund the development of IVF for rhinos.
The move won him fans across the world – fans who will now be mourning his death and the northern white rhino’s proximity to extinction.
Sudan’s genetic material was collected on Monday, conservationists said, to support future attempts to reproduce northern white rhinos through advanced cellular technologies, using stored semen and eggs from the remaining females, Najin and Fatu.
The male rhino, who was the equivalent of 90 in human years, was being treated for degenerative changes in his muscles and bones, combined with extensive skin wounds.
He was unable to stand up and was suffering a great deal in his last 24 hours, Ol Pejeta Conservancy tweeted on Tuesday.