Raphanus sativus L. belongs to the Brassicaceae family, and is known as a traditional medicinal plant. Its seed, Raphani Semen, has certain treatment effects on various diseases such as hypertension, constipation, glycosuria, and asthma. Owing to its great pharmaceutical potential, many chemical ingredients related to its clinical application have been isolated to date. This study isolated two previously unknown and four known 5-thio-4-pentenenitrile derivatives from Raphani Semen through silica gel and preparative HPLC. The two-dimensional (2D) nuclear magnetic resonance spectroscopy (NMR) spectrum and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) identified the two new derivatives as 5-(methylsulfinyl)-4-(methylthio) pentanenitril and (4E)-5-[6-O-(phenylcarbonyl)-β-glucopyranosylsulfanyl]-4-pentenenitrile. Furthermore, the chemical structures of the four known derivatives were elucidated through 1H NMR and 13C NMR and comparison with relevant literature data. The findings improve our understanding of the chemical constituents of Raphani Semen, and may help reveal the mechanism for its clinical application.