Myoblasts characterize by the potential to transform into skeletal muscle, and involve in processes of proliferation, differentiation and apoptosis. Mammalian target of rapamycin (mTOR) is an important protein of PI3K signaling pathway in muscle metabolism and physiology. This study aimed to investigate effects of mTOR on proliferation, apoptosis and differentiation of myoblasts undergoing mechanical stress. We paid much attention on mTOR function undergoing mechanical stress in myoblasts. C2C12 myoblasts were cultured and mTOR gene was knocked down by using Crisper/Cas9 method. Western blot assay and quantitative polymerase chain reaction (Q-PCR) were used to test 4E binding protein 1 (4EBP1) and p70 ribosomal protein S6 kinase (p70S6k) expression. Cell counting kit 8 (CCK-8) was used to measure cell proliferation, and flow cytometry to used to detect cell apoptosis. Differentiation was counted by using immunofluorescence staining. Rresults showed that the knockdown of mTOR reduced the phosphorylation of 4EBP1 and p70S6k levels undergoing mechanical stress and decreased PI3K signaling pathway proteins synthesis. In addition, proliferation of myoblasts was decelerated by the mTOR knockdown. However, when mTOR knocked down cells treated with mechanical stress, apoptosis rate increased significantly and the differentiation speed was slow down. In conclusion, our study revealed the mTOR function on regulating myoblast proliferation, apoptosis and differentiation undergoing mechanical stress.