Xanthohumol (XN), a natural prenylated flavonoid extracted and isolated from the hop plant (Humulus lupulus), possesses diverse pharmacological activities. Although the metabolites of XN have been investigated in the previous study, a comprehensive metabolic profile has been insufficient in vivo or in vitro until now. The current study was aimed at systematically elucidating the metabolic pathways of XN after oral administration to rats. Herein, a UHPLC-Q-Exactive Orbitrap MS was adopted for the potential metabolites detection. A stepwise targeted matching strategy for the overall identification of XN metabolites was proposed. A metabolic net (53 metabolites included) on XN in vivo and in vitro, as well as the metabolic profile investigation, were designed, preferably characterizing XN metabolites in rat plasma, urine, liver, liver microsomes, and feces. On the basis of a stepwise targeted matching strategy, the net showed that major in vivo metabolic pathways of XN in rats include glucuronidation, sulfation, methylation, demethylation, hydrogenation, dehydrogenation, hydroxylation, and so on. The proposed metabolic pathways in this research will provide essential data for further pharmaceutical studies of prenylated flavonoids and lay the foundation for further toxicity and safety studies.