The plant glutathione Stransferase gene (GST) plays an important role in scavenging oxidative damage caused by biotic and abiotic stresses. In order to explore the response mechanism of GST in allelopathic autotoxicity mediated by root exudates, we obtained a GST gene closely related to root exudates mediated autotoxicity in melon by RTPCR based on transcriptome sequencing and using melon leaf DNA as template, named CmGST (GenBank login number: AYU66762.1). In this study, the CmGST gene was analyzed by bioinformatics, and the differential expression of CmGST, the activity of glutathione Stransferase (GST) and the content of glutathione (GSH) in the process of autotoxic stress were determined. The results showed that: (1) the CmGST gene contains a complete open reading frame (ORF) of 654 bp, encoding 217 amino acids. CmGST was an acidic hydrophilic protein and was relatively stable. It has two typical conserved domains of GST family Tau subfamily. CmGST protein had close relationship with Cucummis sativus. (2) Subcellular localization indicated that CmGST was located in the cytoplasm. (3) The results of qPCR analysis showed that CmGST was expressed in root and seedling of melon, and the expression level was relatively stable in normal growth melon seedlings. The expression of CmGST in leaves decreased firstly and then increased rapidly after autotoxicity stress, and the change trend was similar in roots, but the degree was relatively mild. The analysis of GST activity and GSH content showed that the change trend of GST activity in leaves was basically consistent with the change of gene expression of CmGST. In roots, GST activity increased firstly, then decreased, and finally increased. (4) With the increase of stress time, GSH content in roots and leaves increased in varying degrees, but the content in root was lower than that in leaves. The study indicated that the expression level of CmGST in roots and leaves of melon plants changed during the process of autotoxic stress, and CmGST participated in the response to autotoxic stress mediated by root exudates.