Japanese apricot, Prunus mume Sieb. et Zucc., belonging to the Rosaceae family, produces as defensive agents the cyanogenic glycosides prunasin and amygdalin, which are presumably derived from L-phenylalanine. In this study, we identified and characterized cytochrome P450s catalyzing the conversion of L-phenylalanine into mandelonitrile via phenylacetaldoxime. Full-length cDNAs encoding CYP79D16, CYP79A68, CYP71AN24, CYP71AP13, CYP71AU50, and CYP736A117 were cloned from P. mume ‘Nanko’ using publicly available P. mume RNA-sequencing data, followed by 5′- and 3′-RACE. CYP79D16 was expressed in seedlings, whereas CYP71AN24 was expressed in seedlings and leaves. Enzyme activity of these cytochrome P450s expressed in Saccharomyces cerevisiae was evaluated by liquid and gas chromatography–mass spectrometry. CYP79D16, but not CYP79A68, catalyzed the conversion of L-phenylalanine into phenylacetaldoxime. CYP79D16 showed no activity toward other amino acids. CYP71AN24, but not CYP71AP13, CYP71AU50, and CYP736A117, catalyzed the conversion of phenylacetaldoxime into mandelonitrile. CYP71AN24 also showed lower conversions of various aromatic aldoximes and nitriles. The K m value and turnover rate of CYP71AN24 for phenylacetaldoxime were 3.9 µM and 46.3 min(−1), respectively. The K m value and turnover of CYP71AN24 may cause the efficient metabolism of phenylacetaldoxime, avoiding the release of the toxic intermediate to the cytosol. These results suggest that cyanogenic glycoside biosynthesis in P. mume is regulated in concert with catalysis by CYP79D16 in the parental and sequential reaction of CYP71AN24 in the seedling.