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The HD-Zip gene family plays a vital regulatory role in plant growth and development, but its function in plant secondary metabolism remains to be further elucidated. This study therefore investigated the involvement of the HD-Zip gene family in ginsenoside biosynthesis and identified the genes that were highly correlated. Through correlation analysis between gene expression and ginsenoside content, heatmap and co-expression network analyses, and association analysis between Single Nucleotide Polymorphism (SNP) variations and ginsenoside content, the PgHDZ01 gene was identified as the core candidate gene highly associated with ginsenoside biosynthesis. Subsequently, this gene was shown to significantly respond to Methyl Jasmonate (MeJA) treatment and correlated with changes in ginsenoside content. Further functional verification using Virus-Induced Gene Silencing (VIGS) in Panax ginseng (Ginseng) seedlings and gene overexpression in ginseng hairy roots confirmed the positive regulatory role of PgHDZ01 in ginsenoside biosynthesis. Moreover, its overexpression led to a significant increase in the content of ginsenoside Rb1, and its expression was positively correlated with the change in Rb1 content. The results of this study provide a reference for the comprehensive analysis of the functions of the HD-Zip gene family and the regulatory mechanisms of ginsenoside synthesis. The VIGS method used in this study also provides a new approach for the verification of functional genes in ginseng. The results demonstrate the critical role of PgHDZ01 in regulating ginsenoside biosynthesis, deepening our understanding of the underlying mechanisms of ginsenoside biosynthesis and offering a potential candidate gene for molecular breeding applications. Full article