The development of adipose tissue in skeletal muscle is important for improving meat quality. However, it is still unclear how adipocytes grow in the proximity of muscle fibers. We hypothesized that adipocytes would suppress muscle cell growth so as to grow dominantly within muscle. In this study, we investigated the effect of adipocytes on the differentiation of muscle cells in a co-culture system. The fusion index of C2C12 myoblasts co-cultured with 3T3-L1 adipocytes was significantly lower than that of the control. The expression of myogenin and myosin heavy chain in C2C12 muscle cells co-cultured with 3T3-L1 adipocytes was significantly lower than in the control. Furthermore, the expression of Atrogin-1 and MuRF-1 was higher in C2C12 muscle cells co-cultured with 3T3-L1 adipocytes than the control. These results suggest that 3T3-L1 adipocytes suppress the differentiation of C2C12 myoblasts. In addition, 3T3-L1 adipocytes induced the expression and secretion of IL-6 in C2C12 muscle cells. The fusion index and myotube diameter were higher in C2C12 muscle cells co-cultured with 3T3-L1 cells in medium containing IL-6-neutralizing antibody than the control. Taken together, there is a possibility that adipocyte-induced IL-6 expression in muscle cells could be involved in the inhibition of muscle cell differentiation via autocrine.