The male, named Sudan, died at the age of 45 in the Ol Pejeta Conservancy zoo in Kenya on Monday.
He had suffered a series of infections and various age-related health problems.
But his condition deteriorated over the past couple of days, leaving him unable to stand.
Vets were forced to euthanise him to end his suffering.
The rhino was considered an ambassador for his species and had become a symbol for the fight against poaching.
His death leaves just two northern white rhinos on the planet – his daughter Najin and granddaughter Fatu, who remain at Ol Pejeta.
Experts says the only hope for preserving the subspecies now lies in developing IVF techniques using eggs from the two surviving females, stored northern white rhino semen samples from dead males, and southern white rhino surrogate mothers.
Richard Vigne, chief executive of the wildlife reserve, said: “We at Ol Pejeta are all saddened by Sudan’s death.
“He was an amazing rhino, a great ambassador for his species, and will be remembered for the work he did to raise awareness globally of the plight facing not only rhinos, but also the many thousands of other species facing extinction as a result of unsustainable human activity.
“One day, his demise will hopefully be seen as a seminal moment for conservationists worldwide.”
There has been a dramatic increase in rhino poaching over the past decade.
South Africa has lost more than 1,000 rhinos to poachers every year since 2013.
Their horns are trafficked to China and Vietnam for use in purported health tonics and carvings.
Conservationists across the globe have been expressing sadness at Sudan’s death.
Peter Knights, chief executive of anti-poaching charity WildAid, said: “We can only hope that the world learns from the sad loss of Sudan and takes every measure to end all trade in rhino horn.
“While prices of rhino horn are falling in China and Vietnam, poaching for horn still threatens all rhino species.”
There are fewer than 30,000 wild rhinos among five species remaining worldwide.
This includes around 20,000 southern white rhinos, mostly in South Africa, 5,000 black rhinos in southern and eastern Africa, roughly 3,500 Indian one-horned rhinos in Nepal and India, fewer than 100 Sumatran rhinos and an estimated 60 Javan rhinos.
The western black rhinoceros, a subspecies of the black rhinoceros, was declared extinct by the International Union for Conservation of Nature in 2011. Black, Sumatran and Javan rhinos are listed as critically endangered.
“When are we going to understand that we cannot continue to use and abuse wild species without serious consequences?” said Will Travers, president and co-founder of international wildlife charity Born Free.
“Will there be room for non-human life on earth after we have finished?”
Vet Mark Jones, associate director of Born Free, says Sudan’s death should serve as a warning and stressed the importance of efforts to safeguard the survival of the remaining species.