Glyphosate, a commonly used herbicide, has been associated with environmental pollution and potential health risks to aquatic organisms. This study investigated the effects of glyphosate on the muscle metabolism of grass carp (Ctenopharyngodon idellus) following exposure to environmentally relevant concentrations. Over a 14-day exposure period to varying glyphosate levels, significant disruptions were observed in antioxidant capacity and muscle health. These disruptions were evidenced by reductions in total antioxidant capacity (T-AOC), increases in malondialdehyde (MDA) levels, and decreases in activities of glutathione peroxidase (GSH-PX) and catalase (CAT). Furthermore, exposure to glyphosate resulted in a reduction of vitamin E content and an elevation of hormonal levels, suggesting the potential for endocrine disruption. Metabolomics analysis identified 605 distinct metabolites, with notable alterations in amino acid, carbohydrate, and nucleotide metabolism pathways. Specifically, arginine and glutathione metabolisms were severely impacted, with decreases in key amino acids such as glycine and glutathione at higher glyphosate concentrations. Nucleotide metabolism, particularly purine synthesis, was also significantly affected, with reduced levels of deoxyguanosine and other purine-related compounds. The study further investigated the origins of these differential metabolites using the MetOrigin platform, suggesting a potential involvement of the intestinal microbiota in the metabolic response to glyphosate. These findings highlight the multifaceted adverse effects of glyphosate on fish muscle, including oxidative stress and metabolic dysregulation, which may contribute to diminished muscle quality and health risks for aquatic organisms.