The mechanism of multiple enzymes mediated drug metabolism in gut microbiota is still unclear. This study explores multiple enzyme interaction process of typhactyloside (TYP) with gut microbiota and its lipid-lowering pharmacological activity. TYP, with bioavailability of only 2.78%, is an active component of Typha angustifolia L. and Pushen capsules which is clinically treated for hyperlipidemia. The metabolic process of TYP is identified, and key enzymes involved in TYP metabolism are validated through gene knockout and overexpression techniques. Through overexpressing α-rhamnosidase (Rha) in Escherichia coli, TYP is verified to metabolize into isorhamnetin-3-O-neohesperidin (M1) and isorhamnetin-3-O-glucoside (M2) after removing rhamnose through Rha. Besides, knockout of β-glucosidase (Glu) confirms that TYP generates M3 through Glu after removing glucose. Combined with molecular docking, M3 is transformed to generate 3,4-dihydroxyphenylacetic acid (M4), protocatechuic acid (M5), and 3-hydroxyphenylacetic acid (M6) through flavonoid reductase (Flr) and chalcone isomerase (Chi). In conclusion, multiple enzymes involved in TYP metabolism (Rha/Glu→Flr→Chi) are identified. Through in vivo experiments, combined use of M3 and M5 also shows excellent anti-hyperlipidemia efficacy. This is the first study on complex metabolism mechanism and pharmacological activity of natural flavonoids mediated by multiple enzymes, which provide insight to investigate analogous natural products.