When being stressed, plants require a balance between the resistance pathway and metabolism. Glucosinolates (GS) are secondary metabolics that widely exist in Brassicaceae. Glutathione (GSH) not only participates in plant processing reactive oxygen species (ROS) but also directly participates in GS synthesis as a sulfur donor. Therefore, we used transcriptomic to identify antioxidant and GS metabolism responses in GSH-treated pakchoi. Our study elucidated that GSH can be used as priming to improve oxidative resistance and preferentially stimulate the expression of resistance genes such as CAT1 . The reduction in transcription factor expression inhibits the key steps of the GS synthesis pathway. When ROS returned to normal level, the resistance gene decreased and returned to normal level, while GSH restored the gene expression of GS biosynthesis. This work puts forward the mechanism of GSH in regulating the antioxidant system and glucosinolate metabolic pathway, which provides a basis for further study on the relationship between environmental signals and plant metabolism and provides ideas for follow-up research.