This study tested the hypothesis that female rats pups exposed to maternal separation (MatSep), a model of early life stress, display an exacerbated response to diet-induced obesity compared to male rats. Also, we tested whether postnatal treatment with metyrapone (MTP), a corticosterone synthase inhibitor, would attenuate this phenotype. MatSep was performed in WKY offspring by separation from the dam (3 hr/day, postnatal day 2-14). Upon weaning, male and female rats were placed on regular chow (ND, 18% kcal fat) or HFD (60% kcal fat). Non-disturbed littermates served as controls. In male rats, no diet-induced differences in body weight (BW), glucose tolerance, or fat pad weight and morphology were observed between MatSep and control male rats. However, female MatSep rats displayed increased BW gain, fat pad weights and glucose intolerance compared to control rats (p<0.05). Also, HFD increased plasma corticosterone and leptin levels (p<0.05) in female MatSep compared to control rats while insulin and adiponectin levels were similar between groups. Female control and MatSep offspring were treated with MTP (50 µg/g i.p.), 30 minutes prior to the daily separation. MTP treatment significantly attenuated diet-induced obesity risk factors including elevated adiposity, hyperleptinemia and glucose intolerance. These findings show that exposure to stress hormones during early life could be a key event to enhance diet-induced obesity and metabolic disease in female rats. Thus, pharmacological and/or behavioral inflection of the stress levels is a potential therapeutic approach for prevention of early life stress-enhanced obesity and metabolic disease.