BACKGROUND: Chemical castration of male animals is an alternative to surgical castration for inducing azoospermia, consequent sterility. Intra-testicular injection of zinc gluconate has been used for chemical castration in several animal species. However, its application to equine species, such as donkeys, has yet to be reported. This study aimed to evaluate the use of zinc gluconate for the chemical castration of male donkeys and to compare its effectiveness relative to routine surgical castration. For this purpose, investigations of serum testosterone and anti-Müllerian hormone levels, testicular ultrasonographic echogenicity, and histopathological findings were performed. METHODS: Fourteen clinically healthy adult male donkeys were randomly and equally divided into two groups. The donkeys in group I (n = 7) underwent surgical castration. The donkeys in group II (n = 7) received intra-testicular zinc gluconate injections. The donkeys were kept under close clinical observation for 60 days. Abnormalities in donkey behavior and gross alterations in the external genitalia were recorded daily. Serum testosterone and anti-Müllerian hormone (AMH) levels were measured 15 days before the start of the treatment and 15, 30, 45, and 60 days after treatment. The testicles of group II donkeys were evaluated ultrasonographically. At the end of the study, the testes were removed and histologically examined. RESULTS: Serum testosterone levels significantly declined compared to pre-castration levels in surgically castrated donkeys (group I), but donkeys exposed to chemical castration (group II) showed a non-significant reduction in testosterone levels. Donkeys in the surgical group had considerably lower serum AMH levels. In contrast, there was a non-significant (p > 0.05) increase in AMH levels in the chemical group compared with the pre-sterilization level. In addition, ultrasonographic examination revealed that the testicular echo-density had changed, as observed by a few scattered hyperechoic regions throughout the entire testis parenchyma. The histopathological investigation confirmed the presence of necrosis of the spermatogenic epithelium, increased thickness of the basement membrane of the seminiferous tubules, marked interstitial fibrosis, and shrinkage of the seminiferous tubules. Furthermore, syncytial giant cells were present in the lumen of seminiferous tubules and were associated with Sertoli cell vacuolation. Donkeys subjected to chemical castration (group II) had orchitis, as confirmed histopathologically. CONCLUSION: Intra-testicular injection of zinc gluconate resulted in histopathological and ultrasonographic testicular changes in adult male donkeys, which may affect their reproductive potential. However, it did not significantly alter serum testosterone or AMH levels, indicating that it cannot be used as a substitute for surgical castration in male donkeys.