Copper (Cu) is an important plant micronutrient; however, excessive Cu can disturb the protein structure, affect plant growth and development, and pose as a potential human health risk. Glutathione S-transferase (GST) is the key enzyme in glutathione (GSH) synthesis; thus, it plays crucial role in Cu detoxification. Nonetheless, its regulatory mechanisms remain largely unclear. In this study, we identified a Cu-induced glutathione S-transferase 1 (TaGST1) gene in wheat. Yeast one-hybrid (Y1H) between TaGST1 promoter and Cu-stressed wheat leaf cDNA library was performed and screened out TaWRKY74 transcription factor. Their bind were further verified by using another Y1H and luciferase (LUC) assays Functions of TaWRKY74 were further tested by using transiently silence and overexpression methods. Under Cu stress, TaWRKY74 and TaGST1 expression, GST activity, and GSH content were significantly inhibited in transiently TaWRKY74-silenced wheat plants. However, the contents of hydrogen peroxide (H2O2), malondialdehyde (MDA), and Cu were significantly increased in these silenced wheat plants. Further investigation found that transiently ectopic overexpression of TaWRKY74 increased GSH content, whereas decreased MDA content during Cu stress. Notably, exogenous application of GSH could reversed the adverse effects of transiently TaWRKY74-silenced wheat plants during Cu stress. Taken together, our results suggested that TaWRKY74 regulated TaGST1 expression and affected GSH accumulation under Cu stress, and could be useful to ameliorate Cu toxicity for crop food safety.