He eventually could no longer stand and had to be euthanised, conservancy staff said his illness was linked to old age.
Sudan became an icon for endangered animals all over the world when he became the last ever Northern White Rhino.
Ol Pejeta's CEO Richard Vigne says he hopes the death will be seen as a seminal moment for conservationists worldwide.
There are now just two female Northern White Rhinos left in the world.
But not all hope is lost for the species as scientists now work towards developing invitro fertilisation with previously collected sperm from Sudan.
"Sudan's death is a cruel symbol of human disregard for nature and it saddened everyone who knew him," Director of International Projects at Dvur Kralove Zoo, Jan Stejskal.