Abstract: To explore the effects of glyphosate stress on the physiological metabolism of tea plants, the changes in leaf ultrastructure, physiological and biochemical parameters, antioxidant system, and metabolite profiles were systematically investigated under simulated glyphosate exposure at varying concentrations. The results showed that glyphosate stress induced cellular structural damage. As the glyphosate concentration increased, the chlorophyll content declined. The activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione peroxidase (GSH-Px), along with malondialdehyde (MDA) content, generally exhibited a trend of initial increase followed by a decline. The contents of free amino acids, epigallocatechin (EGC), epigallocatechin gallate (EGCG), epicatechin (EC), and epicatechin gallate (ECG) gradually decreased. Similarly, the total content of eight flavonoids showed a decreasing trend. In contrast, the contents of protocatechuic acid, shikimic acid, and gallic acid increased with rising glyphosate levels. These findings showed that glyphosate impairs the antioxidant defense system of tea plants, triggering a defensive response to reactive oxygen species (ROS) accumulation under stress. Furthermore, glyphosate affected tea plant metabolism by inhibiting the key enzymatic activities and reducing metabolic precursors, thereby inhibiting the biosynthesis of secondary metabolites. This study provides a theoretical basis for the rational application of glyphosate in tea gardens and for the breeding of tea varieties with enhanced stress tolerance.