Late-onset hypogonadism (i.e. androgen deficiency) raises the risk for abdominal obesity in men. The mechanism for this obesity is unclear. Here, we demonstrated that hypogonadism after castration caused abdominal obesity in high-fat diet (HFD)-fed, but not in standard diet (SD)-fed, C57BL/6J mice. Furthermore, the phenotype was not induced in mice treated with antibiotics that disrupt the intestinal microflora. In HFD-fed mice, castration increased feed efficiency and decreased fecal weight per food intake. Castration also induced in an increase of visceral fat mass only in the absence of antibiotics in HFD-fed mice, whereas subcutaneous fat mass was increased by castration irrespective of antibiotics. Castration reduced the expression in the mesenteric fat of both adipose triglyceride lipase and hormone-sensitive lipase in HFD-fed mice, which was not observed in the presence of antibiotics. Castration decreased thigh muscle (i.e. quadriceps and hamstrings) mass, elevated fasting blood glucose levels, and increased liver triglyceride levels in a HFD-dependent manner, whereas these changes were not observed in castrated mice treated with antibiotics. The Firmicutes/Bacteroidetes ratio and Lactobacillus species increased in the feces of HFD-fed castrated mice. These results show that androgen (e.g. testosterone) deficiency can alter the intestinal microbiome and induce abdominal obesity in a diet-dependent manner.