Uveitis, a vision-threatening inflammatory disease worldwide, is closely related to resident microglia. Retinal microglia are the main immune effector cells with strong plasticity, but their role in uveitis remains unclear. N6-methyladenosine (m⁶A) modification has been proven to be involved in the immune response. Therefore, we in this work aimed to identify the potentially crucial m⁶A regulators of microglia in uveitis. Through the single-cell sequencing (scRNA-seq) analysis and experimental verification, we found a significant decrease in the expression of fat mass and obesity-associated protein (FTO) in retinal microglia of uveitis mice and human microglia clone 3 (HMC3) cells with inflammation. Additionally, FTO knockdown was found to aggravate the secretion of inflammatory factors and the mobility/chemotaxis of microglia. Mechanistically, the RNA-seq data and rescue experiments showed that glypican 4 (GPC4) was the target of FTO, which regulated microglial inflammation mediated by the TLR4/NF-κB pathway. Moreover, RNA stability assays indicated that GPC4 upregulation was mainly regulated by the downregulation of the m⁶A "reader" YTH domain family protein 3 (YTHDF3). Finally, the FTO inhibitor FB23-2 further exacerbated experimental autoimmune uveitis (EAU) inflammation by promoting the GPC4/TLR4/NF-κB signaling axis, and this could be attenuated by the TLR4 inhibitor TAK-242. Collectively, a decreased FTO could facilitate microglial inflammation in EAU, suggesting that the restoration or activation of FTO function may be a potential therapeutic strategy for uveitis.