During the synthesis of abiraterone acetate bulk drug in some laboratory batches, two unreported impurities were detected by high-performance liquid chromatography analysis at levels ranging from 0.05 to 0.10% according to the United States Pharmacopeia method. The structures of two impurities were characterized and confirmed by NMR and MS, which were proposed to be [3β-acetoxy-16-(3β-acetoxy-androsta-5,16-dien-17-yl)-17-(3-pyridyl)-androsta-5,16-di-ene] and [3β-acetoxy-16-(3β-acetoxy-androsta-5,16-dien-17-yl)-17-androsta-5,16-di-ene]. It was proved that these impurities come into being during the preparation process of penultimate intermediate (abiraterone). The newly developed LC-UV method was used to monitor the impurity profile in the penultimate intermediate (abiraterone), which was validated by its satisfactory specificity, precision, accuracy and sensitivity. The probable origin of the impurity was also discussed.