AIM: To investigate the role of Epac/Rap1 signaling pathway in hypoxia- reoxygenation injury in H9c2 cells, and to explore the mechanism of vitexin regulating the Epac/Rap1 signaling pathway to protect cardiomyocytes from hypoxia- reoxygenation injury. METHODS: The oxygen glucose deprivation (OGD) model was established using H9c2 cardiomyocytes to simulate hypoxia- reoxygenation injury. The experiment was randomly divided into 7 groups: Normal control group, OGD group, OGD+VT group, OGD+8-CPT+VT group, OGD+ESI-09+VT group, OGD+8-CPT+VT+H-89 group, OGD+ESI-09+VT+H-89 group. Cell viability was measured by MTT. LDH was used to detect cell damage. The expression levels of Epac1 and its downstream Rap1-GTP, CaMK II and ERK proteins in H9c2 cells were detected by Western blot. The expression of Epac1 and Rap1 proteins in H9c2 cardiomyocytes was detected by immunofluorescence. The mRNA expression of Rap1 and Epac1 in H9c2 cardiomyocytes was quantitatively determined by real-time PCR. Calcium ion fluorescence probe (Fluo-3 AM) was used to detect intracellular ［Ca2+］i content. The interaction between Epac1 and Rap1 in cells were detected by Co-IP. RESULTS: Compared with the normal control group, after hypoxia for 5 h and reoxygenation for 1 h, the release of LDH, cell viability, Epac1 protein expression, Rap1 activation and RAP1-GTP up-regulation of H9c2 cardiomyocytes in OGD group were significantly increased. VT (10 μmol/L) significantly inhibited the activation of Epac1 in H9c2 cardiomyocytes after OGD, and then inhibited the expression of downstream Rap1 active form Rap1-GTP. In addition, the expression of CaMK II protein was down-regulated, but ERK phosphorylation was increased, and intracellular calcium overload was alleviated. Epac1 agonist 8-CPT could counteract the effect of VT, and Epac1 inhibitor (ESI-09) combined with VT had synergistic effect. PKA inhibitor (Hmur89) had no effect on the expression of Epac1 and its downstream related proteins in cardiomyocytes. CONCLUSION: Hypoxia-reoxygenation can mediate the activation of Epac1/Rap1 signal pathway in cardiomyocytes. VT can protect cardiomyocytes from hypoxia-reoxygenation injury by inhibiting Epac1/Rap1 signal pathway, down-regulating CaMK II protein expression and promoting ERK phosphorylation.