Aim To investigate the effect of overexpression of Bax inhibitor-1(BI-1) gene in cardiomyocytes on ischemia-reperfusion injury and its mechanism. Methods Thirty SD rats were randomly divided into sham operation group (sham group), ischemia-reperfusion group (I/R group), adenovirus control group (Ad-EGFP group), overexpression of BI-1 genome group (Ad-BI-1 group), cyclosporine A group (CsA group). After the establishment of myocardial ischemia-reperfusion injury(MIRI)model in rats, TTC staining was used to observe myocardial infarction area, TUNEL staining was used to observe myocardial apoptosis, ultrastructural changes of myocardial cells were observed under electron microscope; qRT-PCR was used to detect the expression of BI-1 mRNA in the myocardium of rats in each group. The myocardial cells of neonatal rats were isolated and cultured and divided into control group, hypoxia/reoxygenation group (H/R group), adenovirus control group (Ad-EGFP group), overexpression of BI-1 genome group (Ad-BI-1 group) and cyclosporine A group (CsA group) according to different cell treatment. The subcellular localization of BI-1 was detected by immunocytochemistry, the expression of BI-1 protein was detected by Western blot, and the MPTP opening level of myocardial mitochondria was detected by Calcein-AM; and the expression of apoptosis related proteins Bcl-2, Bax, CytC, Caspase-3 and Caspase-9 were detected by Western blot. Results Compared with sham group, myocardial infarct area, apoptosis number, mitochondrial structure damage in I/R group, Ad-EGFP group, Ad-BI-1 group and CSA group increased significantly (P＜0.05), while those in Ad-BI-1 group and CsA group decreased significantly (P＜0.05) compared with I/R group. qRT-PCR results showed that compared with sham group, BI-1 mRNA expression decreased significantly in I/R group, Ad-EGFP group and CsA group, while it increased significantly in Ad-BI-1 group; Subcellular location showed that BI-1 was mainly located in the endoplasmic reticulum of cardiomyocytes, and the fluorescence intensity of Calcein-AM was significantly lower in H/R group, Ad-EGFP group, Ad-BI-1 and CsA group than that in control group, while that of Ad-BI-1 and CsA group was significantly higher than that of H/R group; Compared with control group, the expression level of BI-1 protein was significantly decreased in H/R group, Ad-EGFP group and CsA group, but it was significantly increased in Ad-BI-1 group. Western blot results showed that compared with sham group, the ratio of Bcl-2/Bax decreased significantly in I/R group, Ad-EGFP group, Ad-BI-1 group and CsA group (P＜0.05), the expression of Caspase-3 and Caspase-9 increased significantly (P＜0.05); while the ratio of Bcl-2/Bax increased significantly in Ad-BI-1 group and CsA group compared with I/R group (P＜0.05), and the expression of Caspase-3 and Caspase-9 decreased significantly (P＜0.05). The expression of CytC was significantly higher in I/R group, Ad-EGFP group, Ad-BI-1 group and CsA group than that of sham group (P＜0.05). However, the expression of CytC in the mitochondria of myocardial cells was significantly lower in I/R group, Ad-EGFP group, Ad-BI-1 group and CsA group than that of sham group (P＜0.05), and the expression of CytC was significantly higher in the Ad-BI-1 group and the CsA group than that of I/R group (P＜0.05). Conclusion Overexpression of BI-1 gene can reduce the myocardial infarct area of MIRI rats, reduce the apoptosis of myocardial cells and improve the mitochondrial structure and function damage. The mechanism may be that overexpression of BI-1 can play the above role by changing the Bcl-2/Bax ratio, inhibiting the opening of MPTP, reducing the release of CytC, and reducing the activation of Caspase-3 and Caspase-9.