The in vitro growth inhibitory effects of 27 bufadienolides and eight degradation products, with two cardenolides (ouabain and digoxin) chosen as reference compounds, were analyzed by means of an MTT colorimetric assay in six human and two mouse cancer cell lines. A structure-activity analysis was then performed to highlight the most important substituents relating to the in vitro growth inhibitory activity of bufadienolides in cancer cells. Thus, the current study revealed that various bufadienolides, including gamabufotalin rhamnoside (1a), bufotalin (2a), and hellebrin (3a), displayed higher growth inhibitory activities for various human cancer cell lines when compared to ouabain and digoxin. Gamabufotalin rhamnoside (1a) was the only compound that displayed growth inhibitory effects of <1 μM in mouse cancer cells that expressed mutated forms of the Na(+),K(+)-ATPase α-1 subunit. In addition, all genins and degradation products displayed weaker (if any) in vitro growth inhibitory effects on cancer cells when compared to their respective glycosylated homologue, with the exception of hellebrigenin (3b), which was as active as hellebrin (3a).