Toad venom (venenum bufonis, also called Chan'su) has been widely used for centuries in China to treat different diseases, especially for cancer. Bufadienolides are mainly responsible for the anti-cancer effects of toad venom. However, systematic chemical composition and cytotoxicity as well as key pharmacophores of these bufadienolides from toad venom have not yet been defined clearly. To enrich the understanding of the diversity of bufadienolides and to find bufadienolides with better activities from toad venom. This study was carried out to isolate chemical constituents, research their anti-tumor effects and mechanisms by MTT assay, flow cytometry and Western blotting, and develop a CoMFA and CoMSIA quantitative structure-activity relationship (QSAR) model for illustrating the vital relationship between the chemical structures and cytotoxicities. Among 47 natural bufadienolides, most of bufadienolides (21 compounds isolated in this study and 26 compounds isolated previously) could significantly inhibit the proliferation of cancer cells, and compounds 1, 8, 12, 18 and 19 showed the most potent inhibitory activity against four types of human tumor cells. Compound 18 induced G2/M cell cycle arrest and apoptosis. Moreover, 3D contour maps generated from CoMFA and CoMSIA identified several pharmacophores of bufadienolides responsible for the anti-tumor activities. Our study might provide reliable information for future structure modification and rational drug design of bufadienolides with anticancer activities in medical chemistry. Copyright © 2019 Elsevier B.V. All rights reserved.