Transforming growth factor-beta (TGF-beta) has been demonstrated to inhibit myogenesis in myoblasts. Here we report that transcriptional upregulation of p21(WAF1/Cip1) and muscle creatinine kinase (MCK) genes in C2C12 cells, which are associated with growth arrest at the G1 phase and representative cell-lineage-specific expression during myogenesis, was inhibited by TGF-beta treatment. C2C12 cells expressed TWIST2, but not TWIST1, which was downregulated in myogenic differentiation in response to low-serum cultures. We further found that TGF-beta prevented differentiation-associated downregulation of TWIST2 transcription, resulting in maintaining TWIST2 mRNA expression as high as that in growing C2C12 cells. Ectopic TWIST2 suppressed the p21 and MCK promoters activated by the exogenous addition of E2A and MyoD and this inhibitory effect of TWIST2 was cancelled by the introduction of short hairpin RNA (shRNA) against TWIST2. On the other hand, TWIST2 shRNA failed to recover endogenous promoter activities from TGF-beta-mediated repression. The results clearly indicate that TGF-beta inhibits G1 arrest and maintains TWIST2 expression in C2C12 cells. Our data however suggest that the TGF-beta signaling pathway may not involve TWIST2 to inhibit p21 and MCK expression.