<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>While aging is a potent risk factor of dry eye disease, age-related gut dysbiosis is associated with inflammation and chronic geriatric diseases. Emerging evidence have demonstrated that gut dysbiosis contributes to the pathophysiology or exacerbation of ocular diseases including dry eye disease. However, the relationship between aging-related changes in gut microbiota and dry eye disease has not been elucidated. In this pilot study, we investigated the association between aging-dependent microbiome changes and dry eye severity in C57BL/6 male mice.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>Eight-week-old (8 W, <jats:italic>n</jats:italic> = 15), one-year-old (1Y, <jats:italic>n</jats:italic> = 10), and two-year-old (2Y, <jats:italic>n</jats:italic> = 8) C57BL/6 male mice were used. Dry eye severity was assessed by corneal staining scores and tear secretion. Bacterial genomic 16 s rRNA from feces was analyzed. Main outcomes were microbiome compositional differences among the groups and their correlation to dry eye severity. In aged mice (1Y and 2Y), corneal staining increased and tear secretion decreased with statistical significance. Gut microbiome α-diversity was not different among the groups. However, β-diversity was significantly different among the groups. In univariate analysis, phylum <jats:italic>Firmicutes</jats:italic>, <jats:italic>Proteobacteria,</jats:italic> and <jats:italic>Cyanobacteria</jats:italic>, <jats:italic>Firmicutes</jats:italic>/<jats:italic>Bacteroidetes</jats:italic> ratio, and genus <jats:italic>Alistipes</jats:italic>, <jats:italic>Bacteroides</jats:italic>, <jats:italic>Prevotella</jats:italic>, <jats:italic>Paraprevotella</jats:italic>, and <jats:italic>Helicobacter</jats:italic> were significantly related to dry eye severity. After adjustment of age, multivariate analysis revealed phylum <jats:italic>Proteobacteria</jats:italic>, <jats:italic>Firmicutes</jats:italic>/<jats:italic>Bacteroidetes</jats:italic> ratio, and genus <jats:italic>Lactobacillus</jats:italic>, <jats:italic>Alistipes</jats:italic>, <jats:italic>Prevotella</jats:italic>, <jats:italic>Paraprevotella</jats:italic>, and <jats:italic>Helicobacter</jats:italic> to be significantly associated with dry eye severity.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Our pilot study suggests that aging-dependent changes in microbiome composition are related to severity of dry eye signs in C57BL/6 male mice.</jats:p> </jats:sec>