Background: Although the importance of gender as a key determinant in health and illness has been recognized for a long time, systematic studies of gender differences in medicine are still lacking. We hypothesized that interscapular brown adipocyte tissue (BAT), is not only a key tissue contributing to energy expenditure, but also regulates diet-induced thermogenesis, and may be an ideal target for studying gender differences in obesity development in response to a high fat diet (HFD). Methods: We therefore performed differential proteome analysis of BAT from lean and obese rats of both genders fed a HFD using 2-DE combined with MALDI-TOF-MS. Results: When exposed to a HFD, male rats gained more body weight with increased values of plasma biochemical parameters than did female rats. Among 595 matched spots, 48 differentially expressed identified spots showed significant gender differences, whereas 7 proteins showed no gender differences, but did show a HFD response. Conclusions: Proteomic investigations into gender-dimorphic protein modulation in BAT may provide conclusive results showing higher expression of numerous proteins involved in thermogenesis and fat oxidation as well as lower expression of proteins contributing to fat synthesis in female rats than in male rats.