As a common mycotoxin, deoxynivalenol (DON) contaminates cereal grains and feed in field or during processing and storage. DON elicits a spectrum of adverse effects in animals including anorexia and growth retardation. Especially, the presence of DON has also been detected in muscle, suggesting that DON may has the potential to affect the development of muscle. However, the relevant research is very rare and the molecular mechanism remains unclear. Myoblasts differentiation into multinucleated myotubes is one of the crucial steps of skeletal muscle development. In the present study, we investigated the effects of DON on differentiation of myoblasts using murine C2C12 cells model. The results indicated that DON dose-dependent inhibited the formation of myotubes in C2C12 cells. After performing omics techniques, a total of 149 differentially expressed genes were identified. The expression of cytoskeleton proteins and extracellular matrix (ECM) proteins were downregulated by DON. Furthermore, DON significantly downregulated the expression of integrin αv and integrin β5, leading to inhibition of the ECM-integrin receptor interaction. The focal adhesion kinase (FAK) and phosphorylated forms, ras-related C3 botulinum toxin substrate (RAC) and p21-activated kinases 1 (PAK1) were also downregulated by DON. Taken together, our findings suggest that DON has the potent to affect the differentiation of myoblasts via downregulating of cytoskeleton and ECM-integrin-FAK-RAC-PAK signaling pathway.