Small mammals usually showed physiological and behavioral adaptations to cope with seasonal environmental changing, such as temperature, photoperiod, and food etc. To investigate the physiological and behavioral adaptation strategies in Eothenomys miletus of different areas from Hengduan mountain regions in response to high-fat diet, E. miletus in Jianchuan (JC) and Xianggelila (XGLL) were fed a high-fat (HF) diet for 4 weeks and returned to a low-fat (LF) diet for another 4 weeks, body mass, food intake, resting metabolic rate (RMR), activity behavior, serum leptin levels, hypothalamic neuropeptide expressions and body compositions were measured. The results showed that regions and HF diet affected body mass, food intake and RMR significantly, HF diet increased body mass in E. miletus, while regions had significant effect on activity behavior, but HF diet had not affect activity behavior. Regions and HF diet also showed remarkable effects on leptin and hypothalamus Neuropeptide Y (NPY) expression, and leptin positively correlated with body mass and negatively correlated with NPY expression. Moreover, E. miletus from two regions showed some physiological differences, such as RMR and food intake in XGLL were higher than that of JC, but body mass was lower than that of JC. Body mass reduced quickly inJC after re-feeding LF diet, while it decreased body mass gradually in XGLL. All of the results showed that body mass increased in E. miletus of two regions when faced with HF diet, which returned to the control levels after re-feeding LF diet, showing higher phenotypic plasticity. Leptin and NPY expression may play an important role in body mass regulation. Phenotypic differentiations in E. miletus of two regions may be closely related to food resources, altitude and temperature of Hengduan mountain regions.