PURPOSE: Programmed death ligand 1 (PD-L1) is a potential target for autoimmune disease therapies. The gut microbiota plays a critical role in autoimmunity, and may influence therapeutic outcomes of immune therapies in cancer. However, the relationship between PD-L1 and gut microbiota in autoimmune conditions remains unclear. This study aims to investigate the effect of PD-L1 knockout on gut microbiota in an experimental autoimmune uveitis (EAU) model. METHODS: EAU was induced via immunization with interphotoreceptor retinoid-binding protein peptide 651-670 (IRBP651-670) in either wild type (WT) or PD-L1 knockout (KO) C57BL/6J female mice. Sham adjuvant was administered to WT or PD-L1 KO mice as healthy controls. The severity of EAU was evaluated through clinical evaluation and histopathological gradings. The characteristics of gut microbiota was analyzed using metagenomic sequencing. RESULTS: Each group consisted of three biological replicates. The clinical and histopathological scores of EAU were significantly higher in KO_EAU mice than in WT_EAU mice. WT_EAU mice exhibited lower microbial richness than their healthy controls (WT mice), while PD-L1 KO in EAU mice (KO_EAU group) led to increased richness when compared to wild type EAU mice (WT_EAU group). EAU induced a reduction in the abundance of and an increased in sp. PD-L1 knockout in EAU led to an increased abundance of families Bacteroidaceae, Lachnospiraceae and Ruminococcaceae. EAU was associated with declining microbial tryptophan metabolism and up-regulated functions related to lipid and carbohydrate metabolism; PD-L1 knockout in EAU further increased the metabolism of glycan and biosynthesis of 3-deoxy-α-D-manno-2-octulosonate (Kdo), a key component of bacterial lipopolysaccharide (LPS). CONCLUSION: Both EAU and PD-L1 knockout modulate gut microbiota, affecting microbial composition - particularly , , Bacteroidaceae, Lachnospiraceae and Ruminococcaceae - and microbial functions such as lipid, carbohydrate and glycan metabolism.