Abstract The wintering strategy of the Djungarian hamster (Phodopus sungorus) includes a naturally occurring decrease in food intake and body mass. Our aim was to investigate the conceivable role of the metabolic regulators, AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), in the seasonal adaptation of the Djungarian hamster. In addition, a rate-limiting enzyme in fatty acid synthesis and oxidation, acetyl CoA carboxylase (ACC), was studied. Relative protein expressions and phosphorylated forms (pAMPK and pACC) were determined by Western blot from subcutaneous white adipose tissues (sWAT), abdominal white adipose tissues (aWAT), interscapular brown adipose tissues (iBAT), skeletal muscle, and hypothalamus of winter- and summer-acclimatized hamsters. The winter group had higher AMPK expression in sWAT, aWAT, and iBAT, but the relative amount of phosphorylated protein (pAMPK/AMPK ratio) was lower in these tissues. Furthermore, ACC expression was higher in sWAT and iBAT of the winter animals. pACC (inactive form) levels were higher in all adipose tissues, yet a lower pACC/ACC ratio was detected in iBAT of the winter hamsters. Muscle AMPK expression was lower but pAMPK/AMPK ratio higher in the winter group. SIRT1 expression was higher in muscle and all adipose tissues of the winter hamsters. Hypothalamic protein expressions did not differ between the groups. Higher expressions of AMPK, ACC, and SIRT1 in WAT and iBAT of the winter hamsters suggest a role in the regulation of lipid reserves and increased thermogenic capacity characteristic to the winter-adapted Djungarian hamsters.