Black swallowtail larvae are specialists feeding exclusively on plants containing toxic furanocoumarins and other allelochemicals. Two cytochrome P450s, CYP6B1 and CYP6B3, with an amino acid identity of 87% from black swallowtail were heterologously coexpressed with house fly P450 reductase in a baculovirus/insect cell-mediated expression system and their activities toward a range of ecologically relevant chemicals were subsequently determined. CYP6B1 has the highest activity toward linear furanocoumarins, substantial lower activity toward angular furanocoumarins, and very low activity toward furanochromones and flavonoids. In contrast, CYP6B3 metabolizes the same range of plant allelochemicals tested as CYP6B1 but with no apparent preference for any specific substrates. CYP6B3 activity toward all tested CYP6B1 substrates were at levels comparable to CYP6B1 activity toward angular furanocoumarins. Our results suggest that the more generalized CYP6B3 might have been an evolutionary ancestor for the more specialized CYP6B1. Metabolic data were further explained using molecular modeling and substrate docking analyses.