Objective To observe the expression of fibroblast growth factor 21 (FGF21) for inducing the primary myocardial cells by high glucose and to explore the protection effect of endogenous FGF21 on the cardiomyocyte hypertrophy induced by high glucose. Methods Cardiomyocytes of neonatal rat C57BL/6J were primarily cultured and expressions of FGF21 were detected for different concentrations of glucose (5, 25, 33, and 45 mmol/L) and different exposure time (24 and 48 h). The small interfering RNA (siRNA) for silencing the FGF21 gene was designed and synthesized. Morphological changes of cardiomyocytes were observed and expressions of hypertrophy phenotype ANP and β-MHC mRNA of cardiomyocytes were detected by the Real-time PCR. Results The FGF21 level increased with the concentration of glucose. The FGF21 level of high glucose groups (33 and 45 mmol/L) was higher than that of the control group at the same time point (P<0.05). With the increase of exposure time, the FGF21 level increased first and then decreased. The FGF21 level at 24 h increased and was higher than that at 48 h (P<0.05). The expression of FGF21 mRNA of high glucose groups (33 and 45 mmol/L) was higher than that of the control group. With the increase of exposure time, the expression of FGF21 mRNA increased first and then decreased (P<0.05). After being treated by FGF21-siRNA, the cardiomyocyte hypertrophy induced by high glucose was more significant and expressions of ANP and β-MHC increased (P<0.05). Conclusion The expression of endogenous FGF21 depends on the concentration of glucose to a certain extent under high glucose condition. FGF21 may involve in the process of cardiomyocyte hypertrophy induced by high glucose and protect cardiomyocytes against the hypertrophy.