Autotoxicity is one of the key reasons for serious continuous cropping obstacles in melon cultivation. In this experiment, the aqueous extract of melon plant was used to simulate the autotoxicity stress of melon. The mechanism of melon autotoxicity was investigated by measuring the changes of seed germination, protective enzyme activity and MDA content in root of melon seedlings and transcriptome analyses under autotoxicity stress. The results showed that: (1) the autotoxicity inhibited the germination and subsequent growth of melon seeds, and 0.03 g/mL plant aqueous extract was the threshold value of autotoxicity stress. (2) The activities of protective enzymes and MDA content changed dramatically in root of melon seedlings after treated by melon plant aqueous extracts. SOD activity decreased first, then increased and then decreased. POD activity decreased first and then increased, CAT activity increased first and then decreased and MDA content continued to increase. (3) Transcriptome analysis identified 2 599 differentially expressed genes (DEG) related to melon autotoxicity. There were 2 251 DEGs on 2 d after stress, significantly more than 329 on 4 d. 923 DEGs were upregulated and 1 328 were down regulated in 2 d compared with 0 d. (4) The results showed that melon seedlings had a positive response to autotoxicity at the gene level. Relevant bioinformatics analysis showed that the DEGs caused by autotoxicity were mainly related to phenylpropanoid metabolism, reactive oxygen species (ROS) metabolism, photosynthesis and plant hormone signal transduction, as well as gene differential expression in osmotic regulation, membrane and protein protection. Moreover, these DEGs were mainly enriched in 2 d and 0 d, indicating that photosynthesis of seedlings changed under autotoxicity stress, and this change mainly occurred in the early stage of stress. (5) Six DEGs closely related to melon autotoxicity, AP22, bZIP1, bZIP2, AP21, bHLH and HIS, were analyzed by qRTPCR. The results showed that the expression of AP21, bHLH and HIS reached the peak at 2 d, and AP22 peaked at 2 d in the control group, which were consistent with the results of transcriptome analysis results. The results showed that autotoxicity of melon caused the abnormality of plant cells, and then deteriorated the growth and the formation of organ structure. At the same time, a large number of DEGs related to stimulation or stress were induced. The number of shortterm response genes or transcription factors was significantly higher than that of longterm response under autotoxicity stress, suggesting that melon seedlings can respond quickly to autotoxicity stress.