Inflammation plays an important role in the pathogenesis of diabetic retinopathy. Monocyte chemoattractant protein-1 (MCP-1) is a major inducible chemokine that recruits monocytes and T cells to the sites of inflammation. Upregulation of MCP-1 expression and secretion in the retinal tissue is critical for leukostasis, breakdown of the blood-retinal barrier, and neurovascular injury in diabetic retinopathy. The present study aimed to explore the role of activating transcription factor 4 (ATF4) in MCP-1 regulation in microvascular endothelial cells. Using mouse brain microvascular endothelial cells and retinal endothelial cells, we demonstrated that activation of TLR4 signaling by lipopolysaccharide (LPS) resulted in elevated ER stress and level of ATF4. Overexpression of ATF4 enhanced MCP-1 expression and secretion while inhibition of ATF4 function reduced but not completely reversed TLR4-induced MCP-1 expression. Intravitreal injection of adenovirus expressing ATF4 dramatically increased retinal MCP-1 level, enhanced CD11b expression, and increased leukocyte infiltration into vitreous. In contrast, LPS-induced MCP-1 upregulation in the retina was markedly attenuated in ATF4 knockout mice. Furthermore, monocyte/macrophage migration was enhanced by conditioned media from endothelial cells pretreated with Ad-ATF4 or LPS. Condition media from ATF4 knockout mice induced less monocyte/macrophage migration than that from wild type mice. Mechanistically, our data suggest that ATF4 increases MCP-1 production through enhancing activation of MyD88/NF-KB and AP-1 pathways. Simultaneously, ATF4 binds to the NE-F2 site of MCP-1 promoter. Overexpression of ATF4 up-regulates Nrf2 expression, which in turn suppresses ATF4-induced MCP-1 production. Taken together, we identified a novel regulation and cross-talk loop between ER stress and the TLR4 pathway, in which ATF4 plays an important role in regulation of MCP-1 production and TLR4-mediated endothelial inflammation.