Ginsenosides, as the main pharmacological activity ingredients of ginseng, can be used to treat various diseases. Different cultivars of ginseng have been developed through domestication and artificial selection. However, it is still unclear whether there are differences in the content of ginsenosides and the regulatory mechanisms involved among cultivars. In this study, the roots, stems, and leaves of different ginseng cultivars were analyzed for ginsenosides content and transcriptome data. The results showed that the total ginsenosides content in leaves was higher than that in roots and stems, moreover, the content of total ginsenosides and numerous monomers varied significantly among ginseng cultivars. Among 105 ginsenosides synthesis genes, most of the UDP-glycosyltransferases (UGTs) were highly expressed in roots, while most genes from the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway were overexpressed in leaves. A large number of differentially alternative splicing genes (DASGs) were found in different comparison groups, and the overlapping genes of DEGs and DASGs were rare, furthermore, the functions of DEGs and DASGs were various, suggesting that alternative splicing (AS) and differential expression might play different roles. A series of genes involved in the ginsenosides synthesis showed differentially AS events in the comparison of cultivars. Weighted Gene Co-Expression Network Analysis (WGCNA) was constructed to analyze the gene-ginsenosides association and gene regulatory network. Then, extensive transcription factors (TFs) were identified in highly correlated modules, including AP2/ERF, WRKY, MYB, and bHLH et al., and the expression levels of these TFs were highly correlated with the expression levels of ginsenosides synthesis genes. Therefore, cultivars influenced the structure and expression of ginsenosides synthesis genes through AS and TFs, respectively, thereby modulating the accumulation of ginsenosides. The results provided a theoretical basis for investigating the mechanism of ginsenoside synthesis and offered a new perspective for the development of natural medicines.