Bergenin, a rare C-glycoside with antitussive and expectorant properties, is widely used in China to treat chronic tracheitis. However, its low abundance and structural complexity make it challenging to produce through traditional methods or chemical synthesis. This study elucidates the biosynthetic pathway of bergenin in *Ardisia japonica* by identifying key enzymes, including two 2-C-glycosyltransferases (AjCGT1 and AjCGT2) and four 4-O-methyltransferases (AjOMT2, AjOMT3, AjOMT4, and AjOMT5). The biosynthetic pathway involves the conversion of gallic acid (GA) to 4-O-methyl gallic acid 2-Cβ-D-glycoside (4-OMGA-Glc), which is then esterified to form bergenin. In *Escherichia coli*, the de novo biosynthetic pathway of 4-OMGA-Glc was reconstructed, and the production of bergenin was optimized. The engineered E. coli strain produced bergenin at a titer of 1.41 g L⁻¹ in a 3-L bioreactor, demonstrating the potential for sustainable supply of bergenin through synthetic biology.Bergenin, a rare C-glycoside with antitussive and expectorant properties, is widely used in China to treat chronic tracheitis. However, its low abundance and structural complexity make it challenging to produce through traditional methods or chemical synthesis. This study elucidates the biosynthetic pathway of bergenin in *Ardisia japonica* by identifying key enzymes, including two 2-C-glycosyltransferases (AjCGT1 and AjCGT2) and four 4-O-methyltransferases (AjOMT2, AjOMT3, AjOMT4, and AjOMT5). The biosynthetic pathway involves the conversion of gallic acid (GA) to 4-O-methyl gallic acid 2-Cβ-D-glycoside (4-OMGA-Glc), which is then esterified to form bergenin. In *Escherichia coli*, the de novo biosynthetic pathway of 4-OMGA-Glc was reconstructed, and the production of bergenin was optimized. The engineered E. coli strain produced bergenin at a titer of 1.41 g L⁻¹ in a 3-L bioreactor, demonstrating the potential for sustainable supply of bergenin through synthetic biology.