Abstract: Objective To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes. Methods The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for α-SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca2+ concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting. Results Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein (P<0.05) and increased expressions of CaN (P<0.05) and NFAT3 proteins with also increased intracellular CaN and Ca2 + concentrations (P<0.01). Alda-1 treatment significantly lowered Ca2+ concentration (P<0.05), intracellular CaN content (P<0.01), and CaN and NFAT3 protein expressions (P<0.05), and increased ALDH2 protein expression (P<0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca2 + concentration (P<0.01) and intracellular CaN content (P<0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (P>0.05) but significantly reduced the expression of NFAT3 protein (P<0.05). Conclusion Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca2+-CaNNFAT3 signaling pathway.