Objective To explore the specific injured signal pathways under the condition of hypoxia/reoxygenation (H/R) injury in primary cardiac microvascular endothelial cells (CMEC), and investigate the protective mechanisms of exenatide. Methods Primary CMEC cells were islolated from SD rats with double-enzyme digestion and cultured in vitro. Immunocytochemical staining of CD31 antibody and Ⅷ factors were used to identify the purity of the obtained cells. The injury model of H/R was established. MTT assay was used to detect cell viability for the optimal concentrations of exenatide. The changes of intracellular calcium under H/R condition was detected with the aid of Fluo-3AM fluorescence probe. Western blotting was employed to measure the expression of xanthine oxidase (XO). The level of intracellular reactive oxygen species (ROS) was measured by DCFH-DA labeled ROS probe. Confocal microscopy was performed to observe the JC-1 staining and cytochrome C(Cyt-C) release. After XO siRNA and exenatide pre-treatment were applied to intervent the cells, the above indices were studied again. The measurement data were expressed as mean ±standard deviation ([AKx-D]±s). Student’s t test was used for mutual groups comparison, and one-way ANOVA for multiple groups comparison. Results The optimal anti-apoptotic concentration of exenatide was 10 nmol/L. H/R injury induced the increases in the intracellular calcium level and calcium-dependent XO expression level, while exenatide pre-treatment could decrease the calcium level (P<0.05). Both siRNA and exenatide pre-treatment significantly suppressed the expression of XO, reduced the level of XO-induced ROS, decreased the H/R injury induced nitochrondrial membrane potential, reduced Cyt-C release, and decreased caspase-3 and caspase-9 activities (P<0.05). Conclusion H/R activates Ca²⁺-XO-ROS signaling pathway, leads to mitochondrial structural dysfunction, and finally induces cell apoptosis. exenatide pre-treatment can protect the CMEC cells by suppressing the signaling pathway and reduce apoptosis induced by H/R injury.