OBJECTIVE To investigate the effect of intracavernous injection of human umbilical cord blood derived endothelial colony forming cells (HUCB ECFCs) on erectile dysfunction (ED) in Zucker Diabetic Fatty (ZDF) rat model. METHODS Erectile function was assessed by cavernous nerve electrostimulation in ZDF rats aged 20-28 weeks. Following confirmation of severe ED at the age of 28 weeks, 21 ZDF rats were randomly assigned to three experimental groups: 1 million ECFCs, 2 million ECFCs, and phosphate buffered saline (PBS). Four weeks after intracavernous injection, the efficacy of ECFCs was quantified by intracavernous pressure (ICP) measurement, Masson's trichrome staining, immunohistologic and immunoblot analyses and TUNEL assay. RESULTS Intracavernous ECFC administration improved ICP in a dose-dependent manner in comparison to the age-matched PBS group. Functional improvement in ICP was accompanied by a significant restoration of the cavernosal endothelial and smooth muscle cell content and cavernosal nerve function. The percentage eNOS and nNOS positive cavernosal cells, and their respective protein expression levels and nNOS positive cells in the dorsal penile nerve in 2 million ECFCs treated groups were significantly higher than the PBS group. TUNEL stain quantification showed a significant decrease in cavernosal apoptosis following ECFC treatment. CONCLUSIONS The results are expected to provide a scientific basis to further study the clinical application of HUCB ECFCs in ameliorating ED in human. CONCLUSIONS HUCB ECFCs significantly improved severe ED in ZDF rats through improvement of the nerve and endothelium function and restoration of smooth muscle in the cavernosum by overcoming the cavernosal apoptosis.