D. Don is a medicinal plant rich in 20-hydroxyecdysone (20E), alkaloids, and other active substances. In this study, the cell suspension was incubated for 7 days, followed by the analysis on the effects of abscisic acid (ABA) on the regulation of 20E synthesis. Then suspension cells treated with 0.15 mg/l ABA were used as material, with the Illumina technology applied for transcriptome sequencing. Digital analysis on the gene expression profile was carried out on ABA treated and control samples, respectively. Finally, transcriptomics was applied to assess the molecular response of induced by ABA through applying transcriptomics by evaluating differentially expressed genes. The results suggested that ABA promoted 20E accumulation, while longer processing time caused cell browning. A total of 154 genes were significantly regulated after ABA treatment, with 99 up-regulated and 55 down-regulated, respectively. In addition to 20E-related pathways, the genes belonged to the ko00900 (terpenoid backbone biosynthesis) pathway (six differentially expressed genes [DEGs]), ko00100 (steroid biosynthesis) pathway (four DEGs), and ko00140 (steroid hormone biosynthesis) pathway (six DEGs). Providing a better understanding of the 20E biosynthetic pathway and its regulation, in particular in plants, this study is necessary.