Glyphosate (GLY), as the active ingredient of the most widely used herbicide worldwide, is commonly detected in the environment and living organisms, including humans. Its toxicity and carcinogenicity in mammals remain controversial. Several studies have demonstrated the hepatotoxicity of GLY; however, the underlying cellular and molecular mechanisms are still largely unknown.Using single-cell RNA sequencing (scRNA-seq), immunofluorescent staining, and in vivo animal studies, we analyzed the liver tissues from untreated and GLY-treated mice.We generated the first scRNA-seq atlas of GLY-exposed mouse liver. GLY induced varied cell composition, shared or cell-type-specific transcriptional alterations, and dysregulated cell-cell communication and thus exerted hepatotoxicity effects. The oxidative stress and inflammatory response were commonly upregulated in several cell types. We also observed activation and upregulated phagocytosis in macrophages, as well as proliferation and extracellular matrix overproduction in hepatic stellate cells.Our study provides a comprehensive single-cell transcriptional picture of the toxic effect of GLY in the liver, which offers novel insights into the molecular mechanisms of the GLY-associated hepatotoxicity.© 2023. The Author(s).