Background <p>Uveitis is an autoimmune disease characterized by iris, ciliary muscle, and choroid inflammation. miR-223-3p, an anti-inflammatory microRNA, can regulate the expression of inflammatory genes involved in the disease process. However, the role and potential mechanism of miR-223-3p against uveitis remain unclear.</p> Methods <p>STRING website prediction, molecular docking, and co-IP experiments were performed to verify whether there was an interaction between RBPJ-HDAC1, HDAC1-P65, and P65-ARG1. Based on ex vivo and in vivo experiments, we detected NF-κB P65^lys310 acetylation, P65 nuclear translocation, and the level of M1/M2 macrophage polarization. In addition, we also determined the levels of mitochondrial pressure and calcium flux under different conditions. Regulation of NF-κB P65^lys310 acetylation via the RBPJ/HDAC1 axis affects macrophage polarization and mitochondrial function.</p> Results <p>We first found reduced miR-223-3p expression, elevated RBPJ and total acetylation levels, and an imbalance in macrophage polarization in peripheral blood monocyte-derived macrophages from patients with uveitis. Co-IP experiments supported the interaction between RBPJ and HDAC1, and HDAC1, as a key deacetylase, could inhibit NF-κB P65^lys310 acetylation. Notably, overactivation of NF-κB P65^lys310 acetylation levels in uveitis leads to elevated polarization of M1 macrophages and mitochondrial dysfunction. miR-223-3p can attenuate NF-κB P65^lys310 acetylation and P65 nuclear translocation levels through the RBPJ/HDAC1 axis in uveitis, effectively reducing pro-inflammatory macrophage levels and mitigating mitochondrial damage, thereby reducing ocular inflammation and positively regulating the intraocular microenvironment in uveitis.</p> Conclusion <p>miR-223-3p can inhibit P65^lys310 acetylation and enhance mitochondrial function to improve M1/M2 macrophage polarization balance to ameliorate uveitis through RBPJ/HDAC1 axis.</p> Graphical abstract <p></p>