During cardiac muscle development, most cardiomyocytes permanently withdraw from the cell cycle. Previously, by suppressive subtractive hybridization, we identified calcyclin-binding protein/Siah-interacting protein (CacyBP/SIP) as one of the candidates being upregulated in the hyperplastic to hypertrophic switch, suggesting an important role of CacyBP/SIP in cardiac development. To show the importance of CacyBP/SIP during myoblast differentiation, we report here that CacyBP/SIP is developmentally regulated in postnatal rat hearts. The overexpression of CacyBP/SIP promotes the differentiation and DNA synthesis of H9C2 cells and primary rat cardiomyocytes, as well as downregulates the expression of beta-catenin. Besides, CacyBP/SIP promotes the formation of myotubes and multinucleation upon differentiation. To investigate the cardioprotective role of CacyBP/SIP in cardiomyocytes, a hypoxia/reoxygenation model was employed. We found that CacyBP/SIP was upregulated during myocardial infarction (MI) and hypoxia/reoxygenation. As a conclusion, CacyBP/SIP may play a role in cardiomyogenic differentiation and possibly protection of cardiomyocytes during hypoxia/reoxygenation injury.