Diabetic retinopathy (DR) is a serious-threatening complication of diabetes and urgently needed to be treated. Evidence has accumulated indicating that microglia within retinal plays a critical role in DR. Microglial matrix metalloproteinase 9 (MMP-9) has an important role in destruction the integrity of blood-retinal barrier (BRB) associate with the development of DR. Paeoniflorin, a monoterpene glucoside, has a potent immunomodulatory effect on microglia. We hypothesized that paeoniflorin could significantly suppress microglial MMP-9 activation induced by high glucose and further relieve DR. BV2 cells were used to investigate the effects and mechanism of paeoniflorin. The activation of MMP-9 was measured by gelatin zymography. Cell signaling was measured by western blot assay and immunofluorescence assay .High glucose increased the activation of MMP-9 in BV2 cells, which was abolished by HMGB1, TLR4, p38 MAPK and NF-κB inhibition. Phosphorylation of p38 MAPK induced by high glucose was decreased by TLR4 inhibition in BV2 cells. Paeoniflorin induced HSP70 and SOCS3 expression, and reduced MMP-9 activation in BV2 cells. The effect of paeoniflorin on SOCS3 was abolished by TLR4 inhibitor. In STZ-induced diabetes mice, paeoniflorin induced SOCS3 expression and reduced MMP-9 activation. Paeoniflorin suppressed STZ-induced IBA1 and IL-1β expression, and decreased STZ-induced high blood glucose level. Paeoniflorin induced microglial SOCS3 expression through HSP70/TLR4/NF-κB signaling pathway to reduced MMP-9 in DR prevention.