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<i>Eleutherococcus senticosus </i>(Ruper. et Maxim.) Maxim is a traditional Chinese medicine. The saponin components of <i>E. senticosus</i> have several biological effects, including reduction of blood lipids; protection against liver, heart, and vascular disease; and antitumor activity. The DNA methylation of <i>E. senticosus</i> farnesyl pyrophosphate synthase (<i>FPS</i>), squalene synthase (<i>SS</i>), and squalene epoxidase (<i>SE</i>) gene promoters and the mechanism of the influence of these enzymes on saponin synthesis and accumulation in <i>E. senticosus </i>were explored using bisulfite sequencing technology, real-time PCR, the vanillin-concentrated sulfuric acid chromogenic method, and LC-MS. There are 19 DNA methylation sites and 8 methylation types in the <i>FPS</i> gene. The <i>SS </i>gene has nine DNA methylation sites and two DNA methylation types. The <i>SE</i> gene has 16 DNA methylation sites and 7 methylation types. The total saponin content in the high and low DNA methylation groups were 1.07 &#177; 0.12 and 2.92 &#177; 0.32 mg/g, respectively. Statistical analysis indicated that the gene expression of the <i>FPS</i>, <i>SS,</i> and <i>SE</i> genes was significantly positively correlated with the saponin content (<i>p </i>&lt; 0.05), and that the methylation ratio was significantly negatively correlated with the saponin content (<i>p </i>&lt; 0.01), while the expression of the <i>SS</i> and<i> SE</i> genes was significantly positively correlated (<i>p </i>&lt; 0.01). A total of 488 metabolites were detected from <i>E. senticosus</i> and 100 different metabolites were screened out by extensive targeted metabolomics. The amount of most metabolites related to the mevalonate pathway was higher in the low DNA methylation group than in the high DNA methylation group. It was demonstrated that there are DNA methylation sites in the promoter regions of the <i>FPS</i>, <i>SS,</i> and <i>SE</i> genes of <i>E. senticosus</i>, and DNA methylation in this region could significantly inhibit synthesis in the mevalonate pathway, thus reducing the content of the final product <i>E. senticosus</i> saponin.