Aim To investigate the effect of miR-140-3p on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and its mechanism. Methods In vitro cardiomyocyte H/R model was constructed, and H9c2 cells were transfected with miR-140-3p mimics and chemokine receptor 4 (CXCR4) overexpression plasmids. Cell viability was detected by methyl thiazolyl tetrazolium method. Cell apoptosis was detected by flow cytometry. Reverse transcription polymerase chain reaction and Western blot were used to detect miR-140-3p, CXCR4, and the activation of Janus protein tyrosine kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) pathway and the expressions of apoptosis-related proteins in cells. The targeting relationship between miR-140-3p and CXCR4 was verified by dual-luciferase reporter gene experiment. The activity of lactate dehydrogenase (LDH) and the levels of inflammatory factors and reactive oxygen species (ROS) were detected with corresponding kits. Results In vitro H/R could inhibit the expression of miR-140-3p, up-regulate the expression of CXCR4 and the phosphorylation of JAK2/STAT3 pathway, induce the apoptosis of H9c2 cells and inhibit the proliferation of H9c2 cells, promote the release of inflammatory factors and ROS, and up-regulate the activity of LDH. miR-140-3p could inhibit the expression of CXCR4 by targeting CXCR4 3′UTR, thereby inhibiting the phosphorylation activation of JAK2/STAT3 pathway, inhibiting the release of inflammatory factors and ROS, down-regulating the activity of LDH, promoting the proliferation of H9c2 cells and inhibiting their apoptosis. Conclusion miR-140-3p may inhibit the JAK2/STAT3 pathway through targeted inhibition of CXCR4, thereby attenuating ischemia/reperfusion-induced cardiomyocyte injury.