Blood-retinal barrier (BRB) dysfunction has been recognized as an early pathological feature in common eye diseases that cause blindness. The breakdown of endothelial cell-to-cell junctions is the main reason for BRB dysfunction, yet our understanding of junctional modulation remains limited. Here, we demonstrated that endothelial-specific deletion of TBK1 (Tbk1(Delta EC)) disrupted retinal vascular development, and induced vascular leakage. LC-MS/MS proteomic analysis was used to identify candidate substrates of TBK1. We found that TBK1 interacted with CXCR4, and the phosphorylation level of CXCR4-Serine 355 (Ser355) was decreased in Tbk1(Delta EC) retina samples. Furthermore, TBK1-mediated phosphorylation of CXCR4 at Ser355 played an indispensable role in maintaining endothelial junctions. Interestingly, we also detected an increased expression of TBK1 in diabetic retinopathy samples, which suggested an association between TBK1 and the disease. Taken together, these results provided insight into the mechanisms involved in the regulation of endothelial cell-to-cell junctions via TBK1-dependent CXCR4 phosphorylation.