Objective: To investigate the effect of Ginkgolide B (GKB) on human retinal endothelial cell (HREC) injury induced by high concentration of glucose and hypoxia. Methods: The high glucose and hypoxia-induced HREC injury cell model were established, then the optimal GKB concentration and time point were selected by CCK-8 assay. The inflammatory reaction was evaluated by the levels of TNF-α, ICAM-1 and IL-6 which were detected with ELISA. Cell apoptosis was determined by flow cytometry. Finally, activation of PI3K/AKT/mTOR pathway was detected with Western blot. Results: Compared with the normal control group, cells in high glucose and hypoxia model group have a lower cell viability, and GKB could reverse the effect of high glucose and hypoxia. High glucose and hypoxia led to a rise of early cell apoptotic ratio and higher expression of TNF-α, ICAM-1 and IL-6; however, after treatment with GKB, cell apoptotic ratio and the expression level of TNF-α, ICAM-1 and IL-6 decreased compared with high glucose and hypoxia model group. Furthermore, high glucose and hypoxia down-regulated the protein expression of p-AKT, p-PI3K and p-mTOR; while compared with high glucose and hypoxia model group, the protein expression of p-AKT, p-PI3K and p-mTOR were partly increased, after treatment with GKB. Incubation with IGF-1 (AKT pathway agonist) could promote the activation effect of GKB on PI3K/AKT/mTOR pathway and up-regulate cell viability. Conclusion: Ginkgolide B could remarkably alleviate high glucose and hypoxia-induced HREC apoptotic injury and inflammatory reaction, and its protective effect may be related to the activation of PI3K/AKT/mTOR pathway.