Objective The goal of this study is to understand the function of FKBP51 in resistant to high fat diet-induced obesity using FKBP51 knockout (KO) mice and in vitro adipocyte differentiation. Methods Four-week old male FKBP51 KO and wild type (WT) mice were fed separately with regular or high fat diet for 6 weeks. The body weight and food consumption were recorded weekly, the energy expenditure differences (O₂ consumption, CO₂ production, respiratory exchange ratio, and heat production) of each group were monitored using the MM-100 metabolism cages system for 24 hours, then the liver from the above animals were stained with the Oil red-O to detect the lipid accumulation and the expression of metabolic genes. In addition, induction of adipocyte differentiation of immortalized MEF cells from WT and FKBP51 KO mice were used to observe the effect of FKBP51 gene on lipogenesis. Results Compared to WT mice, FKBP51 KO mice has less weight increment, and less lipid accumulation in the liver, but with no difference on food consumption during high-fat diet fed. Moreover, FKBP51 KO mice exhibited more O₂ consumption, CO₂ production and heated production under both RD and HF diet conditions. The PEPCK,G6Pase and UCP-1 genes up-regulation. In addition, lipid content was reduced in FKBP51 gene deficient MEF cells after adipocyte differentiation. Conclusions The FKBP51 gene plays an important role in high fat diet-induced obesity through the energy metabolism enhancement and lipogenesis inhibition.