Canstatin, a cleaved fragment of type IV collagen α2 chain, is highly expressed in myocardium of normal rats. We previously demonstrated that full length canstatin exerts an anti-hypertrophic effect in several rat cardiac disease models. However, the active sites of canstatin have not been determined. In this study, we explored the active sites of canstatin, which could exhibit protective effects against angiotensin II (Ang II)-induced cardiac hypertrophy. Full length and N-terminal (1-82 amino acids: N-canstatin) but not central (83-149 amino acids) or C-terminal (150-220 amino acids) fragment of canstatin inhibited Ang II-induced hypertrophy in neonatal rat cardiomyocytes. In a rat model of Ang II-induced hypertension, N-canstatin did not affect an increase in systolic blood pressure, while it showed a tendency to inhibit an increase in heart weight/tibia length ratio and significantly suppressed cardiomyocyte hypertrophy. N-canstatin significantly inhibited myocardial fibrosis and tended to suppress mRNA expression of type I collagen in left ventricles. In the present study, we for the first time demonstrate that N-terminal fragment of canstatin has protective effects against hypertrophic cardiac remodeling.