AbstractBackground and PurposePathological retinal angiogenesis is a typical manifestation of vision‐threatening ocular diseases. Many patients exhibit poor response or resistance to anti‐vascular endothelial growth factor (VEGF) agents. Bruton's tyrosine kinase (BTK) controls the proliferation and function of immune cells. Therefore, we examined the anti‐inflammatory and anti‐angiogenic effects of BTK inhibition on retinal angiogenesis.Experimental ApproachRetinal neovascularisation and vascular leakage in oxygen‐induced retinopathy in C57/BL6J mice were assessed by whole‐mount retinal immunofluorescence. PLX5622 was used to deplete microglia and Rag1‐knockout mice were used to test the contribution of lymphocytes to the effects of BTK inhibition. The cytokines, activation markers, inflammatory and immune‐regulatory activities of retinal microglia/macrophages were detected using qRT‐PCR and immunofluorescence. NLRP3 was detected by western blotting, and the effects of BTK inhibition on the co‐culture of microglia and human retinal microvascular endothelial cells (HRMECs) were examined.Key ResultsBTK inhibition suppressed pathological angiogenesis and vascular leakage, and significantly reduced retinal inflammation, which involved microglia/macrophages but not lymphocytes. BTK inhibition increased anti‐inflammatory factors and reduced pro‐inflammatory cytokines that resulted from NLRP3 inflammasome activation. BTK inhibition suppressed the inflammatory activity of microglia/macrophages, and acted synergistically with anti‐VEGF without retinal toxicity. Moreover, the supernatant of microglia incubated with BTK‐inhibitor reduced the proliferation, tube formation and sprouting of HRMECs.Conclusion and ImplicationsBTK inhibition suppressed retinal neovascularisation and vascular leakage by modulating the inflammatory activity of microglia and macrophages. Our study suggests BTK inhibition as a novel and promising approach for alleviating pathological retinal angiogenesis.LINKED ARTICLESThis article is part of a themed issue Drugs and Drug Targets in Metabolic and Chronic Inflammatory Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.20/issuetoc