Objective To investigate the effect of high fat diet feeding on mitochondrial structure and function. Methods Male C57BL/6J mice at the age of 4 weeks were used in this study. After 6 weeks of regular diet (RD) or high-fat diet (HF) feeding, the high fat-induced obesity phenotype was confirmed by body weight measurement and liver histopathology. RNA was isolated from the liver tissue of RD and HF mice and the expression profiles were detected using RNA-seq. Differentially expressed genes between RD and HF mice were analyzed using BRB-ArrayTools. DAVID online tools were applied to analyze the GO and KEGG pathways. Transmission electron microscopy and western blotting were performed to observe the mitochondrial ultrastructure and quantified the expression of function-related proteins. Results Compared with the RD mice, the body weight gain was faster in the HF mice. The size of the lipid droplets was bigger in the HF-fed mouse liver tissue. Multiple pathway analysis all identified that these major gene changes were related to mitochondria. The mitochondrial deformation, enlarged or even destruction was observed in the high fat diet group observed by transmission electron microscopy. This observation was further confirmed by detecting of the expression of genes in the HF liver mitochondria. The levels of MFN1 and PHB1 were significantly increased, while the level of FKBP51 was significantly decreased. Conclusions FKBP 51 is involved in the high-fat-induced mitochondrial damage via morphological and structural damages of mitochondria.