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Ginseng (<i>Panax ginseng</i> C. A. Meyer) is an ancient and valuable Chinese herbal medicine, and ginsenoside, as the main active ingredient of ginseng, has received wide attention because of its various pharmacological active effects. Cytochrome P450 is the largest family of enzymes in plant metabolism and is involved in the biosynthesis of terpenoids, alkaloids, lipids, and other primary and secondary plant metabolites. It is significant to explore more <i>PgCYP450</i> genes with unknown functions and reveal their roles in ginsenoside synthesis. In this study, based on the five <i>PgCYP450</i> genes screened in the pre-laboratory, through the correlation analysis with the content of ginsenosides and the analysis of the interactions network of the key enzyme genes for ginsenoside synthesis, we screened out those highly correlated with ginsenosides, <i>PgCYP309</i>, as the target gene from among the five <i>PgCYP450</i> genes. Methyl jasmonate-induced treatment of ginseng adventitious roots showed that the <i>PgCYP309</i> gene responded to methyl jasmonate induction and was involved in the synthesis of ginsenosides. The <i>PgCYP309</i> gene was cloned and the overexpression vector pBI121-PgCYP309 and the interference vector pART27-PgCYP309 were constructed. Transformation of ginseng adventitious roots by the <i>Agrobacterium fermentum</i>-mediated method and successful induction of transgenic ginseng hairy roots were achieved. The transformation rate of ginseng hairy roots with overexpression of the <i>PgCYP309</i> gene was 22.7%, and the transformation rate of ginseng hairy roots with interference of the <i>PgCYP309</i> gene was 40%. Analysis of ginseng saponin content and relative gene expression levels in positive ginseng hairy root asexual lines revealed a significant increase in PPD, PPT, and PPT-type monomeric saponins Re and Rg2. The relative expression levels of <i>PgCYP309</i> and <i>PgCYP716A53v2</i> genes were also significantly increased. <i>PgCYP309</i> gene promotes the synthesis of ginsenosides, and it was preliminarily verified that <i>PgCYP309</i> gene can promote the synthesis of dammarane-type ginsenosides.