The widespread use of glyphosate, a broad-spectrum herbicide, has resulted in significant human exposure, and recent studies have challenged the notion that glyphosate is safe for humans. Although the link between disease states and glyphosate exposure is increasingly appreciated, the mechanistic links between glyphosate and its toxic effects on human health are poorly understood. Recent studies have suggested that glyphosate may cause toxicity through modulation of the gut microbiome, but evidence for glyphosate-induced gut dysbiosis and its effect on host physiology at doses approximating the U.S. Acceptable Daily Intake (ADI = 1.75 mg/kg body weight) is limited. Here, utilizing shotgun metagenomic sequencing of fecal samples from C57BL/6 J mice, we show that glyphosate exposure at doses approximating the U.S. ADI significantly impacts gut microbiota composition. These gut microbial alterations were associated with effects on gut homeostasis characterized by increased proinflammatory CD4⁺IL17A⁺ T cells and Lipocalin-2, a known marker of intestinal inflammation.