Rhizoma coptidis, the root of Coptis chinensis Franch, has been used in China as a folk medicine in the treatment of diabetes for thousands of years. Berberine, one of the active ingredients of Rhizoma coptidis, has been reported to improve symptoms of diabetes and to treat experimental cardiac hypertrophy, respectively. The objective of this study was to evaluate the potential effect of berberine on cardiomyocyte hypertrophy in diabetes and its possible influence on peroxisome proliferator-activated receptor- α (PPAR α )/nitric oxide (NO) signaling pathway. The cardiomyocyte hypertrophy induced by high glucose (25.5 mmol/L) and insulin (0.1 μ mol/L) (HGI) was characterized in rat primary cardiomyocyte by measuring the cell surface area, protein content, and atrial natriuretic factor mRNA expression level. Protein and mRNA expression were measured by western blot and real-time RT-PCR, respectively. The enzymatic activity of NO synthase (NOS) was measured using a spectrophotometric assay, and NO concentration was measured using the Griess assay. HGI significantly induced cardiomyocyte hypertrophy and decreased the expression of PPAR α and endothelial NOS at the mRNA and protein levels, which occurred in parallel with declining NOS activity and NO concentration. The effect of HGI was inhibited by berberine (0.1 to 100 μ mol/L), fenofibrate (0.3 μ mol/L), or L-arginine (100 μ mol/L). MK886 (0.3 μ mol/L), a selective PPAR α antagonist, could abolish the effects of berberine and fenofibrate. N (G) -nitro-L-arginine-methyl ester (100 μ mol/L), a NOS inhibitor, could block the effects of L-arginine, but only partially blocked the effects of berberine. These results suggest that berberine can blunt HGI-induced cardiomyocyte hypertrophy in vitro, through the activation of the PPAR α /NO signaling pathway.