Abstract: Two cardenolide glycosides, corotoxigenin 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-β-D-glucopyranoside ( 1 ) and coroglaucigenin 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-β-D-glucopyranoside ( 2 ), were isolated from the seed fairs of Asclepias curassavica. The structures of 1 − 2 were determined based on the combination of the analysis of their MS, NMR spectroscopic data and acid hydrolysis. The inhibitory effects of compounds 1 and 2 on human colorectal carcinoma cells (HCT116), non-small cell lung carcinoma cells (A549) and hepatic cancer cells (SMMC-7721) were evaluated. The results showed that both compounds 1 and 2 significantly inhibited the viability, proliferation, and migration of A549, HCT116 and SMMC-7721 cells, suggesting that compounds 1 and 2 can be applied in the treatment of lung, colon and liver cancers in clinical practice. This study may not only provide a scientific basis for clarifying the active ingredients in A. curassavica, but also help to understand its antitumor activity, which can promote the application of A. curassavica in clinical treatment of various cancers.