【Objective】To explore the effect of histone H3K27me3 methyltransferase Ezh2 deficient in mesenchymal stem cells（MSC）on the differentiation of mouse skeletal muscle cells and mitochondria inside them. 【Methods】Ezh2 conditional knockout mice（Ezh2F/FPrx1-Cre）were constructed. In 6-week-old mice，HE staining，modified Gomori trichrome staining and NADHTR staining were used to observe cell morphology and mitochondria. The ultrastructures of skeletal muscle cells and mitochondria were observed under electron microscope. Western blot was used to detect the protein expression of Ezh2，H3K27me3，Myogenin，Myosin，Desmin and CytC. The expression of differentiation related genes and mitochondrial related genes in skeletal muscle were detected by Realtime RT-qPCR. The expression of CytC in skeletal muscle was detected by immunohistochemistry，and ATP content in skeletal muscle was detected by ATP content assay kit. HE staining and NADHTR staining were used to observe the skeletal muscle of 2-week-old mice. In 6-week-old mice，the content of Ezh2 protein in brown adipose tissues was detected by Western blot，and the mitochondria were observed under electron microscope.【Results】The content of Ezh2 and H3K27me3 protein in the skeletal muscle of the knockout group decreased significantly. In the knockout group，the muscle fibers of skeletal muscle were not uniform in thickness；some nuclei moved inward，and skeletal muscle markers Myogenin and MhcIIb mRNA expression decreased significantly（P < 0.05）. Myogenin protein expression decreased，but NADH-TR and CytC immunohistochemical staining showed that mitochondria increased. Under electron microscope，the myofibrils in the knock-out group were of different thickness，and the number of mitochondria under the cell membrane and between myofibrils increased significantly， which were clumped. There was no edema in mitochondria and no obvious decrease in cristae. The expression of CytC， Tfam，Pgc1α mRNA and CytC protein increased in the knockout group（P < 0.05），but the content of ATP decreased significantly（P < 0.05）. However，there was variation in muscle fiber thickness but no difference in the content of NADH of 2-week-old mice. Under electron microscope，there was no increase in the number or abnormal morphology of mitochondria in brown adipocytes of 6-week- old mice.【Conclusions】Ezh2 is necessary in the differentiation of mouse skeletal muscle cells in MSC stage and maintain the ATP production of mitochondria. In Ezh2 deficient mice，the ATP production of mitochondria decreased and the number of mitochondria increased compensatorily.