Cardiac glycosides are commonly used drugs in clinical medicine. We analyzed the cytotoxic effect of six steroids belonging to the bufadienolide family on malignant T lymphoblasts and normal peripheral blood mononuclear cells (PBMC). One compound was a natural bufadienolide glycoside (hellebrin) with cardiac activity. The other five compounds were chemically modified derivatives that did not contain cardioactive groups. We found that these steroids were able to cause time-dependent apoptosis in Jurkat T lymphoblasts, whereas they only minimally affected PBMC. Preferential killing of malignant cells was induced by the natural cardioactive substance hellebrin and by three of the five chemically modified non-cardioactive derivatives. The substances caused mitochondrial transmembrane potential disruption and internucleosomal DNA fragmentation in tumor cells. The cytoplasmic and nuclear events of bufadienolide-induced apoptosis were strongly inhibited in the presence of caspase 8, caspase 9, or caspase 3 inhibitors, as well as in the presence of the broad-spectrum caspase inhibitor Z-VAD-FMK. Overexpression of Bcl-2 significantly protected bufadienolide-treated cells from phosphatidylserine translocation, transmembrane potential disruption, and internucleosomal DNA fragmentation. Our results show that the analyzed bufadienolide derivatives preferentially kill malignant human lymphoblasts by initiating apoptosis via the classical caspase-dependent pathway. Apoptosis-inducing agents specific for tumor cells might be ideal anti-tumor drugs. The therapeutic use of bufadienolides has been hampered by their concomitant cardiac activity. The description of compounds without cardiac activity but with tumor-specific cytotoxicity suggests the potential of using them in cancer therapy.