Objective To investigate the protective effect of total flavonoids in puerarin (PR) on oxidant injury in endothelial cells (ECs) induced by hydrogen peroxide (H₂O₂) and the possible mechanism. Methods ECs were isolated from the aorta of male Sprague-Dawley rats by adherent method. Cell viability was assessed by MTT colorimetry, and the effect of PI3K inhibitor LY294002 on the proliferation was also measured. The cell apoptosis was detected with flow cytometry. Fluorescence microscopy was used to observe the autophagic marker, microtubule-associated protein 1 light chain 3 (LC-3) in the 3 groups of cells. Western blotting was employed to detect the expression of PI3K and AKT in presence or absence of LY294002. One-way ANOVA was used to make compar-ison between groups. Results The cell viability was significantly lower in the H₂O₂₊PR ECs with LY294002 treatment than those without[(2.07±0.08) vs (1.69±0.04), P<0.05]. Flow cytometry showed that the apoptosis rate was significantly higher in the H₂O₂ injured cells and the injured cells with PR treatment than the control cells (P<0.05), but PR treatment reduced the apoptosis rate in the H₂O₂ cells (13.26% vs 25.47%, P<0.05). Compared with the control cells, the total intracellular LC3-GFP fluorescence intensity was enhanced in the H₂O₂ ECs and the H₂O₂₊PR ECs (P<0.05), with that of the latter group more significant (P<0.05). The protein levels of PI3K and AKT were up-regulated significantly in H₂O₂₊PR ECs than the control cells (P<0.05), while the increases were blocked by LY294002 treatment (P<0.05). Conclusion PR protects ECs from H₂O₂ induced injury and promotes cell survival through inhibiting cell excessive apoptosis and autophagy, which may be associated with up-regulation of PI3K/AKT expression.