As a traditional Chinese medicine for reinforcing kidney, Cuscutae semen can be applied in regulating the level of estrogen. And nine direct-acting substances in vivo with estrogenic effect has been figured out according to our previous research. However, their transformation relationship and the prototype forms of some metabolites are still not clear, impeding the elucidation of the effective substances of Cuscutae semen. In this study, to reveal the metabolic pathways of nine direct-acting substances in vivo, the hepatic and intestinal metabolism of nine monomer components from Cuscutae semen were investigated using liver microsomes of female rats and intestinal flora in ovariectomized rats. Based on ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOFMS) method, a total of 35 hepatic metabolites and three intestinal metabolites were tentatively identified respectively. The proposed metabolic pathways of main metabolites were discussed. Glucuronidation binding reactions played a dominant role in the hepatic metabolism. While in the intestinal metabolism, flavonoid glycosides were hydrolyzed into the aglycones without further phase II metabolic reaction. According to the achievements above, a reverse traceability analysis of the direct-acting substances in vivo was conducted, and the metabolic transformation relationship between the components was clarified except for methylated quercetin glucuronide. It is tentatively speculated that kaempferol, quercetin, apigenin, hyperin, astragalin and isoquercitrin in Cuscutae semen are potential estrogenic effective substances, which can be used as reference for quality control.