This study elucidates the biosynthetic pathway of bergenin in Ardisia japonica and reconstructs its de novo synthesis in Escherichia coli. Bergenin, a rare C-glycoside with pharmacological properties, is used in traditional Chinese medicine but is limited by its low abundance in nature. The research identifies key enzymes, 2-C-glycosyltransferases (AjCGT1 and AjCGT2) and 4-O-methyltransferases (AjOMT2 and AjOMT3), involved in its biosynthesis. These enzymes catalyze the formation of 4-O-methyl gallic acid 2-C-β-D-glycoside, the direct precursor of bergenin. The pathway is reconstructed in E. coli, where the precursor is efficiently esterified to bergenin. Through metabolic engineering, the production of bergenin is increased to 1.41 g/L in a 3-L bioreactor. This work provides a sustainable method for the large-scale production of bergenin, addressing its resource scarcity. The study also highlights the potential of synthetic biology in the production of rare natural products. The findings contribute to the understanding of the biosynthetic pathway of C-glycosides and offer a framework for the artificial biosynthesis of bergenin. The research demonstrates the feasibility of using bacterial fermentation systems to enhance the yield of rare C-glycosides through metabolic engineering. The efficient and green production of bergenin from glucose lays a foundation for its supply.This study elucidates the biosynthetic pathway of bergenin in Ardisia japonica and reconstructs its de novo synthesis in Escherichia coli. Bergenin, a rare C-glycoside with pharmacological properties, is used in traditional Chinese medicine but is limited by its low abundance in nature. The research identifies key enzymes, 2-C-glycosyltransferases (AjCGT1 and AjCGT2) and 4-O-methyltransferases (AjOMT2 and AjOMT3), involved in its biosynthesis. These enzymes catalyze the formation of 4-O-methyl gallic acid 2-C-β-D-glycoside, the direct precursor of bergenin. The pathway is reconstructed in E. coli, where the precursor is efficiently esterified to bergenin. Through metabolic engineering, the production of bergenin is increased to 1.41 g/L in a 3-L bioreactor. This work provides a sustainable method for the large-scale production of bergenin, addressing its resource scarcity. The study also highlights the potential of synthetic biology in the production of rare natural products. The findings contribute to the understanding of the biosynthetic pathway of C-glycosides and offer a framework for the artificial biosynthesis of bergenin. The research demonstrates the feasibility of using bacterial fermentation systems to enhance the yield of rare C-glycosides through metabolic engineering. The efficient and green production of bergenin from glucose lays a foundation for its supply.