Gender differences in obesity stem from metabolic and hormonal differences between sexes and contribute to differences between women and men in health risks attributable to obesity. We hypothesized that liver may be an ideal target for the evaluation of gender differences in obesity development in response to a high‐fat diet (HFD). Therefore, to test this hypothesis, we performed a global proteome analysis in the liver of lean and obese rats of both genders who were fed an HFD through 2‐DE combined with MALDI‐TOF‐MS. When rats were exposed to HFD, male rats gained more body weight with increased values of plasma biochemical parameters than female rats. Image analysis and further statistical analysis of a 2‐DE protein map allowed for the detection and identification of 34 proteins that were significantly modulated in a gender‐dependent manner. We found 19 proteins showing identical gender‐different regulation in both normal diet (ND) and HFD. Five proteins also showed clear gender differences in both ND and HFD; however, their regulation modes in HFD were opposite to those in ND. Of particular interest, 10 proteins showed gender differences only in either ND or HFD rats. Present proteomic insight into gender‐dimorphic protein modulation in liver would aid in the improvement of gender awareness in the health‐care system and in implementation of evidence‐based gender‐specific clinical recommendations.