The aim of the study is to explore the effects and mechanism of the action of ligustrazine on isoprenaline-induced cardiomyocyte hypertrophy. Primary culture of neonatal rat cardiomyocytes was used as the model, and isoprenaline was used to induce cardiomyocyte hypertrophy. Effects of different dosages of ligustrazine polysaccharide on the cardiomyocyte were observed. RT-PCR was used to detect the expression of atrial natriuretic factor (ANP) mRNA, and Western blot analysis was used to detect the CaN protein level in cardiomyocytes. After treating with ligustrazine, the significant increase of MDA content and decrease of SOD activity were inhibited in supernatant. Compared to the control group, ANP mRNA in isoprenaline-treated cardiomyocytes was significantly increased (P < 0.05); compared to the isoprenaline group, ANP mRNA was significantly decreased in all ligustrazine groups (P < 0.01). In all ligustrazine groups, the CaN expression was inhibited in isoprenaline-treated cardiomyocytes in a dose-dependent manner. In conclusion, ligustrazine has protective effects on isoprenaline-induced neonatal rat cardiomyocyte, which may be related to the decrease of CaN expression.