Glycyrrhiza inflataBatalin is a medicinal licorice species that has been widely used by humans for centuries. Licochalcone A (LCA) is a characteristic flavonoid that accumulates inG. inflataroots with high economical value. However, the biosynthetic pathway and regulatory network of its accumulation remain largely unknown. Here we found that a histone deacetylase (HDAC) inhibitor nicotinamide (NIC) could enhance the accumulation of LCA and total flavonoids inG. inflataseedlings.GiSRT2, a NIC-targeted HDAC was functionally analyzed and its RNAi transgenic hairy roots accumulated much more LCA and total flavonoids than its OE lines and the controls, indicating a negative regulatory role ofGiSRT2in the accumulation of LCA and total flavonoids. Co-analysis of transcriptome and metabolome of RNAi-GiSRT2lines revealed potential mechanisms in this process. AnO-methyltransferase gene,GiLMT1was up-regulated in RNAi-GiSRT2lines and the encoded enzyme catalyzed an intermediate step in LCA biosynthesis pathway. Transgenic hairy roots ofGiLMT1proved thatGiLMT1is required for LCA accumulation. Together, this work highlights the critical role ofGiSRT2in the regulation of flavonoid biosynthesis and identifiesGiLMT1as a candidate gene for the biosynthesis of LCA with synthetic biology approaches.