Vascular inflammation is characteristic feature of diabetic retinopathy. In diabetic retina, a variety of the pro‐inflammatory cytokines are elevated and involved in endothelial dysfunction. STAT3 transcription factor has been implicated in mediating cytokine signaling during vascular inflammation. However, whether and how STAT3 is involved in the direct regulation of the endothelial permeability is currently undefined. Our studies revealed that IL‐6‐induced STAT3 activation increases retinal endothelial permeability and vascular leakage in retinas of mice through the reduced expression of the tight junction proteins ZO‐1 and occludin. In a co‐culture model with microglia and endothelial cells under a high glucose condition, the microglia‐derived IL‐6 induced STAT3 activation in the retinal endothelial cells, leading to increasing endothelial permeability. In addition, IL‐6‐induced STAT3 activation was independent of ROS generation in the retinal endothelial cells. Moreover, we demonstrated that STAT3 activation downregulates the ZO‐1 and occludin levels and increases the endothelial permeability through the induction of VEGF production in retinal endothelial cells. These results suggest the potential importance of IL‐6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy. J. Cell. Physiol. 232: 1123–1134, 2017. © 2016 Wiley Periodicals, Inc. Vascular inflammation is characteristic feature of diabetic retinopathy. We demonstrated that STAT3 in retinal endothelial cells plays a causative role in downregulating ZO‐1 and occludin levels and increasing the endothelial permeability through VEGF production in retinal endothelial cells. These results suggest the potential importance of IL‐6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy.