Diabetic retinopathy (DR) represents a major complication of diabetes, and molecular mechanisms related to vascular dysfunction, particularly endothelial dysfunction, in DR remains unclear. In the present work, we generated a DR animal model using mice and a cell model in mouse retinal microvascular endothelial cells (mRMECs) to examine the role of Trefoil factor family 1 (Tff1) in DR. Tff1 was poorly expressed in DR mice and high glucose (HG)-treated mRMECs. Overexpression of Tff1 significantly attenuated streptozotocin-induced retinal proliferation and angiogenesis in DR mice and reduced the secretion of inflammatory factors. In HG treated mRMECs, overexpression of Tff1 remarkably reduced the proliferation and angiogenesis of mRMECs. In further experiments, we found that Tff1 was transcriptionally repressed by Runt-related transcription factor 1 (Runx1) directly, and Tff1 expression was indirectly modulated by Runx1 via the core-binding factor subunit beta (CBF-beta)/nuclear factor, erythroid 2/microRNA-423-5p axis and the CBF-beta/estrogen receptor 1 (ESR1) axis. Moreover, Tff1 could inhibit the activation of NF-kappa B signaling pathway, which in turn attenuated retinal endothelial cell proliferation and angiogenesis. It was thus proposed that Runx1/Tff1/NF-kappa B axis may be a potential target for the treatment strategy of DR, and further studies are needed.