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Metabolomics is an implement for testing the toxicity of antibiotics, and provides a comprehensive view of the overall response to stress; however, the connections between metabolites and biologic endpoints keep unclear in response to antibiotics. In this study, wheat seeds were exposed to tetracycline for 5 days. The results proved that tetracycline restrained growth, reduced chlorophyl and carotinoid contents and cell permeability, and increased reactive oxygen species (ROS) levels and malondialdehyde (MDA) content. Orthogonal partial least squares (OPLS) was used to analyze the connections between metabolites and biologic endpoints, which discovered that 11 metabolic pathways were significantly affected by tetracycline, and amino acid metabolism could largely apply to root growth and ROS accumulation, while carbohydrate metabolism could have a ruling effect on tetracycline-induced cell permeability. 13 metabolites all played active roles in mediating tetracycline’s effects on root length, root fresh weight and cell permeability but had no significant effects on ROS levels. The majority of metabolites with passive effects on root length, root fresh weight and cell permeability had active effects on ROS levels. These results offer a view about stress reaction of wheat to tetracycline.