Myogenesis, the formation of muscle fibers, is affected by certain glycoproteins, including chondroitin sulfate (CS), which are involved in various cellular processes. We aimed to investigate the mechanism underlying CS-E-induced suppression of myotube formation using the myoblast cell line C2C12. Differentiated cells treated with 0.1 mg/ml CS-E for nine days showed multinucleated and rounded myotubes with myosin heavy chain positivity. No difference was found between the CS-E-treated group with rounded myotubes and CS (−) controls with elongated myotubes in the levels of phospho-cofilin, a protein involved in the dynamics of actin cytoskeleton. Interestingly, N-cadherin, which is involved in the gene expression of myoblast fusion factors (myomaker and myomixer), was significantly downregulated at both the mRNA and protein levels following CS-E treatment. These results suggest that N-cadherin downregulation is one of the mechanisms underlying the CS-E-induced suppression of myotube formation.