Objective To evaluate the effects of hydrogen peroxide (H 2O 2) on cardiomyocytes and the protective effect of pinacidil preconditioning on injured primary cultured cardiomyocytes. Methods We isolated and cultured cardiomyocytes in neonatal mice by trypsin digestion technique. The cells were inoculated in 96-well culture plate at the density of 1?10 5/ml. Cells were divided into control group and 400, 600, and 800 ?mol/L H 2O 2 treatment groups. MTT assay was used to determine the cell vitality at 1, 3, 6, 12, and 24 h after H 2O 2 treatment. The effects of pinacidil preconditioning were also observed. Results The effect of H 2O 2 on the damages to cardiomyocytes was in dose- and time-dependent manners. Pretreatment with pinacidil at the concentrations of 1?10 -4 , 1?10 -5 , and 1?10 -6 mol/L, particularly 1?10 -6 mol/L, could increase the activity of cardiomyocytes treated with H 2O 2. Conclusion Pinacidil preconditioning at lower doses can protected cardiomyocytes from injuries induced by H 2O 2.