Prohibitin-1 (PHB1), an evolutionarily conserved mitochondrial chaperone, has an important role in adipocyte differentiation. Recently, our laboratory has developed a novel transgenic mouse named “Mito-Ob” by overexpressing PHB1 in adipocytes under the adipocyte protein-2 gene promoter. Mito-Ob mice develop obesity due to upregulation of adipocyte mitochondrial biogenesis in a sex-neutral manner, but only male mice develop impaired glucose homeostasis and insulin resistance. The female Mito-Ob mice are protected from these abnormalities. Sex steroid hormones, estradiol and testosterone, play pivotal roles in body metabolism including sex differences in energy balance and fat distribution. Based on the above information, the present study investigates the relationship between sex steroid hormones and PHB1 on sex differences in Mito-Ob phenotype and in adipocyte functions. Data from both in vitro and in vivo experiments suggest that PHB1 and sex steroid hormones regulate each other’s function in the growth of adipose tissue and subsequently in the regulation of adipose tissue function in a sex dimorphic manner. Overexpression of PHB1 in adipocytes further amplifies sex differences in adipose tissue function suggesting a role of PHB1 in mediating sex dimorphic effects of sex steroid hormones in adipose tissue biology. Furthermore, results suggest that intrinsic differences in adipose tissue exist between male and female independent of sex steroid hormones, which play a role in sex differences in adipose tissue function and consequently on whole body metabolic homeostasis.