Secondary structure of silk fiber containing spider (Araneus ventricosus) fiber protein produced by transgenic silkworm (KH29) was analyzed by 13C CP/MAS NMR and compared with that of wild-type silk fiber. The NMR spectrum showed that the secondary structure of KH29 silk were the same as that of wild-type silkworm silk even though spider fiber protein existed in silk. In addition, since the peak signal around 15.7 ppm, indicating the α-helix of Ala Cβ, was not observed in the NMR spectrum of KH29 silk, in KH29 silk may form a β-sheet structure instead of an α-helix structure after spinning process of silkworm. These results indicate that physical properties of silk can be modified by the incorporation of foreign fiber proteins by the use of transgenic silkworm technology.