The 45-year-old rhino named Sudan had been in poor health in recent days and was being treated for age-related issues and multiple infections.
A veterinary team made the decision to euthanize Sudan after his condition deteriorated significantly, the conservation group WildAid announced Tuesday.
Sudan lived in the Ol Pejeta Conservancy in Kenya, surrounded by armed guards in the days leading up to his death to protect him from poachers.
"He was a gentle giant, his personality was just amazing and given his size, a lot of people were afraid of him. But there was nothing mean about him," said Elodie Sampere, a representative for Ol Pejeta.
Researchers were able to save some of Sudan's genetic material in the hopes of successfully artificially inseminating one of the two females left, Sampere 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," said WildAid CEO Peter Knights.
Rhinos are targeted by poachers, fueled by the belief in Asia that their horns cure various ailments. Experts say the rhino horn is becoming more lucrative than drugs.
In addition to round-the-clock security, the Ol Pejeta Conservancy also put radio transmitters on the animals and dispatched incognito rangers into neighboring communities to gather intelligence on poaching.
Old and frail
At 45, Sudan was elderly in rhino years and suffered from problems associated with age.
During his final years, he was not able to naturally mount a female and suffered from a low sperm count, which made his ability to procreate difficult.
His daughter Najin, 28 and granddaughter, Fatu, considered young by comparison. Najin could conceive, but her hind legs are so weak she may be unable to support a mounted male.
Sudan made headlines last year when the Tinder dating app named him the "most eligible bachelor in the world" in a campaign to raise funds to save the subspecies.
The western black rhino was declared extinct seven years ago as a result of poaching. All five remaining rhino species worldwide are considered threatened, according to the conservation group Save the Rhino.
Correction: This story has been updated to clarify that Sudan was a northern white rhino.