Abstract: To explore the key factors involved in toxin synthesis by the transcriptome sequencing analysis of the retroauricular glands and abdominal skin of Bufo bufo gargarizans at five different developmental stages, it provides a theoretical basis for further study on the regulation mechanism of toxin synthesis. Transcriptome sequencing was performed on the abdominal skin and retroauricular gland of Bufo bufo gargarizans at 5 developmental stages, including 3-month-old, 6-month-old, 12-month-old, 24-month-old, and 36-month-old. The function of differentially expressed genes was analyzed by GO and KEGG. The key genes were screened by weighted co-expression network analysis. The transcriptome data of the abdominal skin and retroauricular gland at the same developmental stage were compared, and 3053, 3 026, 1 516, 1 028, and 2 061 differentially expressed genes were screened, respectively. According to the GO classification statistics, the differentially expressed genes were mainly concentrated in biological processes categories such as cellular processes, monomer processes, metabolic processes, and biological regulations. In the molecular function categories, it is mainly concentrated in the binding, catalytic activity, and so on. In cellular component categories, it is mainly concentrated in the cell, component and organelle. The analysis of the KEGG pathway showed that the differentially expressed genes were involved in many pathways during toad development, such as tyrosine metabolism, peroxisome metabolism, and primary bile acid metabolism. Comparing common differentially expressed genes from 5 developmental stages, twenty-one genes related to steroid metabolism, primary bile acid metabolism, and the peroxisome were identified. Five hub genes and one key transcription factor from four modules were screened by weighted co-expression network analysis. A large number of genes of the metabolic pathway were enriched in the differentially expressed genes between the skin and retroauricular glands of Bufo bufo gargarizans at different developmental stages. These genes may play an important role in regulating the synthesis of retroauricular adenotoxins. This study lays the foundation for understanding the molecular mechanism of toad toxin production.