Flavonoids are phenolic secondary metabolites commonly occurring in plants. In particular, prenylated flavonoids exhibit a wide range of biological activities, including antitumor, antibacterial and antioxidant activities. In this study, we attempted to produce prenylated flavonoids in tomato as a host plant by means of metabolic engineering through the introduction of both the naringenin 8-dimethylallyltransferase (N8DT) gene, encoding a prenyltransferase from Sophora flavescens, and the chalcone isomerase (CHI) gene from Nicotiana tabacum cv. Samsun-NN. Liquid chromatography-tandem mass spectrometry analysis revealed the production of 8-dimethylallyl naringenin in the double transformants, while the production level was lower than N8DT single transformants. In addition, tomato fruits over-expressing both N8DT and CHI genes accumulated high levels of rutin compared with wild-type tomato. A possible endogenous regulation of the synthesis of flavonoid derivatives is discussed.