Abstract Selenium (Se) plays an important role in cancer-prevention. Silkworm pupas have been used as a Chinese traditional medicine since ancient time. In order to find effective carcinostatic agents, Se-rich amino acids were extracted from Ziyang silkworm pupas. The Se content of Ziyang pupas was measured to be 215 times higher than that of Luoyang normal ones, and the majority of Se was stored in proteins. Composition analysis showed that Se-rich amino acids from Ziyang pupas had higher amounts of selenomethionine, methionine, cystine, and tyrosine than normal amino acids from Luoyang pupas which were rich in amino acids containing alkyl side chains. When cultured with human hepatoma cells SMMC-7721, Se-rich amino acids at concentrations of 0.5, 1.5, and 2.5 μmol L − 1 Se significantly and dose-dependently inhibited cell viability, induced changes in cell morphology and cycle, and caused cell apoptosis. On the contrary, normal amino acids did not show any inhibitory effect on SMMC-7721 cells. Sodium selenite or selenomethionine at the same Se concentrations only slightly inhibited the hepatoma cells. Mechanism study showed that selenium-rich amino acids could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. Those results suggested that Se-rich amino acids were effective carcinostatic agents compared with sodium selenite and selenomethionine. The mechanism for their hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation.