Autophagy plays various roles at different stages of ischemia reperfusion (I/R) injury in cardiomyocytes. It has been reported that tissue factor pathway inhibitor (TFPI) has a protective effect on I/R injury. This study aimed to determine the roles of TFPI in autophagy during the I/R injury process in cardiomyocytes and the possible mechanisms. An isolated hypoxia/reoxygenation (H/R) pattern of cardiomyocytes was established by the MIC101 system. The cell viability and oxidative stress of cardiomyocytes were detected by an MTT assay and ROS assay, respectively. The autophagy level was measured by Ad-mCherry-GFP-LC3B and MDC. We detected the expression levels of autophagy-related proteins by western blotting. After 2 h of hypoxia and 12 h of reoxygenation, the cardiomyocyte viability in the H/R group was significantly lower than that in the control group (p < 0.05) than in the H/R group. According to intracellular ROS production, the fluorescence intensity in the H/R group was enhanced compared with that in the negative control group, and it was weaker in the H/R + rTFPI group compared with the H/R group. The level of autophagy and the expression levels of autophagy-related proteins (LC3-II/LC3-I, Beclin-1 and PI3K) were markedly increased in the H/R group compared to the control group (p < 0.05) whereas the levels were markedly decreased in the H/R + rTFPI group compared to the H/R group (p < 0.05). TFPI could relieve cardiomyocyte injury by inhibiting the Class III PI3K/Beclin-1 pathway and oxidative stress; thus, TFPI decreased autophagy and protected cardiomyocytes induced by H/R injury. In conclusion, TFPI may be a new direction for the prevention of myocardial I/R injury.