Aim To investigate the effects of tissue factor pathway inhibitor (TFPI) on cardiomyocyte apoptosis following ischemia/reperfusion (I/R) injury. Methods I/R model was established by ligating the anterior descending coronary artery in vivo. Rats were randomly divided into three groups:the control, I/R, and I/R+rTFPI groups. HE staining was used to evaluate the morphological changes of rats myocardial tissue after reperfusion for 3 days. TTC staining was used to detect the myocardial infarct size. Ultrastructural damage in cardiomyocytes was measured by transmission electron microscopy. The expression levels of Bcl-2, Bax and cleaved Caspase-3 in rats myocardial tissue were detected by Western blot analysis. Cardiomyocytes of Sprague Dawley (SD) rats were cultured by enzyme digestion and differential adherent method in vitro experiments. Cardiomyocytes I/R injury in vitro model was established by 2 h of hypoxia and 12 h of reoxygenation using MIC101 system. Cardiomyocytes were divided into control group, H/R group, and H/R+rTFPI group (10 μg/L). Cell viability was detected by CCK-8 assay. Detection of cardiomyocytes apoptosis was performed by TUNEL technique. Western blot analysis was used to detect the expression of Bax, Bcl-2 and cleaved Caspase-3. Results A myocardial I/R model was successfully established in vivo. HE staining showed myocardiumin exhibited a higher degree of necrosis than that in control group, which was milder in I/R+rTFPI group than that in I/R group (P＜0.05). TTC staining showed that, compared with I/R group, myocardial infarct size reduced 39.76% in I/R+rTFPI group (P＜0.05). The data of transmission electron microscopy showed that the degree of apoptosis and injury was severer in I/R group than that in control group, whereas it was milder in I/R+rTFPI group than that in I/R group. Western blot detection results showed that compared with control group, the expression of Bcl-2 decreased 53.43% in I/R group(P＜0.05), but the expression of Bax and cleaved Caspase-3 increased 29.05% and 73.25% respectively (P＜0.05). After adding rTFPI, the expression of Bax and cleaved Caspase-3 in myocardial tissue decreased 13.77% and 24.25% respectively compared with I/R group(P＜0.05), whereas the expression of Bcl-2 increased 55.01% compared with I/R group (P＜0.05). In in vitro experiment, the CCK-8 results showed the cell viability of H/R group decreased 29.70% compared with control group(P＜0.05), but it increased 19.77% after adding rTFPI compared with H/R group(P＜0.05). The TUNEL detection showed that the apoptotic rate of H/R group increased 56.76% compared with control group, whereas the apoptotic rate of H/R+rTFPI group decreased 24.55% compared with H/R group(P＜0.05). Western blot results showed that compared with control group, the expression of Bcl-2 in cardiomyocytes decreased 46.92% in H/R group (P＜0.05), but the expression of Bax increased 41.90%(P＜0.05) and the expression of cleaved Caspase-3 increased 2.68 fold (P＜0.05). After adding rTFPI, the expressions of Bax and cleaved Caspase-3 in the cardiomyocytes decreased 26.34% and 57.60% respectively when compared with H/R group(P＜0.05), whereas the expression of Bcl-2 increased 28.24% compared with H/R group (P＜0.05). Conclusion TFPI served a protective role against I/R and H/R-induced cardiomyocyte injury by inhibiting apoptosis.