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Introduction: Prevalence of chronic hypohydration remains elevated among adults in the USA; however, the health effects of hypohydration in regards to human gut health have not been explored. Methods: This study examined the relationship between total water intake, hydration biomarkers (first-morning urine specific gravity [FMUsg], first-morning urine volume [FMUvol], and plasma copeptin), fecal microbiota, and plasma lipopolysaccharide-binding protein (LBP) in adults (25–45 years, 64% female). Fecal microbiota composition was assessed using 16S rRNA gene sequencing (V4 region). Immunoassays quantified plasma copeptin and LBP in fasted venous blood samples. Dietary variables were measured using 7-day food records. Linear discriminant analysis effect size (LEfSe) analyzed differentially abundant microbiota based on median cutoffs for hydration markers. Multiple linear regressions examined the relationship between LBP and copeptin. Results: LEfSe identified 6 common taxa at the genus or species level that were differentially abundant in FMUsg, total water (g/day), or plasma copeptin (µg/mL) groups when split by their median values. Uncultured species in the Bacteroides, Desulfovibrio, Roseburia, Peptococcus, and Akkermansia genera were more abundant in groups that might indicate poorer hydration status. Multivariate linear analyses revealed a positive relationship between plasma copeptin and LBP when controlling confounding variables (F(6,52) = 4.45, p = 0.002, R2 = 0.34). Conclusions: Taxa common between markers are associated with the intestinal mucus layer, which suggests a potential link between hydration status and intestinal mucus homeostasis. The relationship between LBP and copeptin indicates that copeptin may be sensitive to metabolic endotoxemia and potentially gut barrier function. [ABSTRACT FROM AUTHOR]