Endocrine system of hormones is the critical factor for the development of testes. The levels of hormones are orchestrated by a positive or negative feedback system controlled by the hyphothalamic-pituitary-gonad axis. The aim of this study was to investigate the effect of unbalanced endocrine system induced by the hemi-castration on testicular development in stallions. Four Thoroughbred stallions (age ranging from 3 to 5 yr) were used in this study. To disturb endocrine system, hemicastration has been performed on the stallions. Several parameters including testicular weight, volume, germ cell population on the cross-sections of round tubule, and the area of seminiferous tubules of stallion testes collected at the 1st hemi-castration and the 2nd hemi-castration (about 1 year after 1st hemi-castration) were compared. Testosterone levels were compared for 3 weeks before, after 1st castration, and before 2nd castration using enzyme-linked immunosorbent assay (ELISA) analysis. Immunohistochemistry (IHC) procedure was conducted to compare germ cell populations between after 1st and 2nd castration using VASA antibody. The VASA positive cell population per a cross section of round seminiferous tubule was obtained by monitoring 100 tubules. Interestingly, the weight of testes obtained at 2nd hemi-castration (384±14 g) were significantly higher compared to that of testes collected at the 1st hemi-castration (288±34 g). The volume of testes (306±34 ml) collected at the 2nd hemi-castration was higher than that of testes (169±18 ml) collected at the 1st castration. In contrast, VASA positive germ cell population on the cross section of round tubule (124.9±12.4 vs 142.9±21.6) and the area of round tubule (124±9.7 vs 122.9±1.7 mm2) were not different after 1st castration and 2nd castration. the testosterone levels in the blood collected before, after 1st castration, and before 2nd castration were not significantly different. In conclusion, the hemi-castration induces testicular development to maintain the normal reproductive systems in stallions.