〔Abstract〕 Objective To investigate protective effect and mechanism of vitexin on rat primary cardiomyocytes induced by hypoxia/reoxygenntion injury via regulating autophagy and autophagic flow. Methods Hypoxia/reoxygenation(H/R) of primary cardiomyocytes was used to simulate ischemia-reperfusion injury. The viability of cardiomyocytes was tested by CCK8. LDH levels in cardiomyocyte supernatant were detected by kit. ROS levels were detected by DCFH-DA fluorescent probe. Primary cardiomyocytes were infected with mRFP-GFP-LC3, and the number of Autophagolysosomes and autophagosomes was evaluated. Beclin1, LC3Ⅱ/Ⅰ and P62 protein expression were detected by Western blot. Results After hypoxia and reoxygenation(H/R) of primary cardiomyocytes, the LDH value of primary cardiomyocytes in the H/R group was significantly higher than that in the control group, and the ROS level significantly increased, the doses of vitexin could significantly inhibit the release of LDH from H/R cardiomyocytes and reduce the ROS level in cardiomyocytes. The primary cardiomyocytes were transfected with mRFP-GFP-LC3 virus, and the results showed that compared with the control group, the cardiomyocytes in the H/R group showed significant enhancement of autophagy and autophagy flow. suggesting that autophagosomes accumulated in myocardial cells after H/R, and autophagolysosomes increased accordingly; The vitexin dose groups could inhibit autophagy and increase autophagy flow in cardiomyocytes after H/R, and reduce the accumulation of autophagosomes in cardiomyocytes. In addition, Western blot results showed that compared with the normal control group, the expression of autophagy proteins Beclin 1, and LC3Ⅱ in primary myocardial cells of the H/R group increased, and the ratio of LC3Ⅱ/Ⅰincreased, P62 protein in cardiomyocytes was down regulated; Compared with the H/R group, vitexin inhibited Beclin 1 up-regulation and decreased the expression of LC3Ⅱ protein, the ratio of LC3Ⅱ/Ⅰreduced, and the expression of P62 protein also increased in cardiomyocytes. Conclusion Vitexin has a significant protective effect on H/R injury in rat primary myocardial cells, and its mechanism may be related to the inhibition of ROS production, autophagy and autophagic flow enhancement, and the accumulation of autophagosomes after H/R in myocardial cells.