Accumulating evidence suggests that caspase-3-mediated cleavage of protein kinase could be a key event to regulate cell differentiation. In this study, we investigated the role of Nek5 kinase, identified as a novel substrate for caspase-3, in skeletal muscle differentiation. Up-regulation of Nek5 mRNA expression was accompanied by cell differentiation. Myotube formation was promoted in Nek5 expressing cells, and was conversely inhibited in Nek5 knockdown cells. Furthermore, we found that caspase-3 activity, an important factor for myogenic differentiation, was enhanced by Nek5 cleavage. Although caspase-3-cleaved Nek5 partially exerted a promyogenic effect, it tended to induce apoptotic cell death. In summary, our findings suggest that Nek5 promotes myogenic differentiation through up-regulation of caspase activity.