Coumarins are a group of natural compounds commonly found in the families of Rutaceae and Umbelliferae. 7-Isopentenyloxycoumarin (ISC), auraptene (AUR), and umbelliprenin (UM) belong to prenyloxycoumarins (PYCs), which link isopentenyl, geranyl, and farnesyl group at C7 position, respectively. The substituent of 7-ethoxycoumarin (ETC) is the ethyl group. In this study, UPLC-ESI-QTOF-MS (ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-MS)-based metabolomics was used to evaluate the in vivo and in vitro metabolism of PYCs. Results showed that ETC produced 10 known metabolites, and ISC was transformed into 17 metabolites in vivo and in vitro, which were undescribed compounds. A total of 35 AUR metabolites, including 34 undescribed metabolites were identified, and 21 metabolites were reported for the first time in UM. The results indicated that hydroxylation and N-acetylcysteine conjugation were the common metabolic reactions for PYCs. The metabolic rates of ETC, ISC, AUR and UM were 26%, 36%, 81%, and 38%, respectively, in human liver microsome, while they were 24%, 40%, 80%, and 37%, respectively, in mouse liver microsomes. In addition, recombinant cytochrome P450s (CYPs) screening showed that CYP1A1, 2C19, 3A4, and 3A5 were the major metabolic enzymes involved in the formation of hydroxylation metabolites. Together, these results suggest that the isopentenyl group plays an important role in the metabolism of PYCs.