ABSTRACT: Glyphosate (GLY) is one of the most used active substances in broad-band herbicides worldwide. Although farm animals might be orally exposed to this herbicide via GLY contaminated feedstuffs, not much is known about possible hepatotoxic effects of GLY. As hepatic xenobiotic and nutrient metabolism are closely interlinked, the toxic effects of GLY residues might be influenced by nutrient supply to the liver. Therefore, a feeding trial with lactating dairy cows was conducted to investigate effects of GLY contaminated feedstuffs and different concentrate feed proportions (CFP) in the diets as a tool for varying nutrient supply to the liver. Therefore, 61 German Holstein cows (207 ± 49 days in milk; mean ± standard deviation) were either fed a GLY contaminated total mixed ration (TMR, GLY, average mean GLY intake 122.7 µg/kg bodyweight/day) or control TMR (CON, average mean GLY intake 1.2 µg/kg bodyweight/day) for 16 weeks. Additionally, both groups were further split into subgroups fed a lower (LC, 30% on dry matter basis) or a higher (HC, 60% on dry matter basis) CFP. Blood parameters aspartate aminotransferase, γ-glutamyltransferase, glutamate dehydrogenase, cholesterol, triglyceride, total protein, calcium, phosphorus, acetic acid and urea as well as histopathological evaluation were not influenced by GLY, whereas all mentioned parameters were at least affected by time, CFP or by an interactive manner between time and CFP. Total bilirubin blood concentration was significantly influenced by an interaction between GLY and CFP with temporarily elevated concentrations in GLYHC, whereas the reasons and biological relevance remain unclear. Gene expression analysis indicated 167 CFP-responsive genes, while seven genes showed altered expression in GLY groups compared to CON groups. Since expression changes of GLY-responsive genes were low and liver-related blood parameters changed either not at all or only slightly, the tested GLY formulation was considered to have no toxic effects on the liver of dairy cows.