The current study was conducted to compare the cytotoxicity of two stereospecific cephalostatin 1 analogues (CAs) against several human normal cell types and cancer cell lines and to determine their cytotoxic mechanism. Both CA analogues induced apoptosis and were cytotoxic with 50% growth inhibition (GI50) at ˜1 μM or less in six human cancer cell lines but neither analogue at 10 μM killed more than 14% of any of three types of normal human cells suggesting their cytotoxicity is cancer-specific. CA treatment inhibited clonogenic tumor growth and activated caspase 3 and 9 but not caspase 8. CA-induced apoptosis was inhibited by the pan caspase inhibitor indicating the importance of caspase activation. CA treatment released smac/DIABLO but not cytochrome c from mitochondria and induced phosphorylation of eIF-2 and the activation of procaspase 4 in cancer cells, similar to cell treatment with thapsigargin, a known endoplasmic reticulum (ER) stress inducer. Finally, cells pretreated with a caspase 4 inhibitor were resistant to CA-induced apoptosis. In conclusion, both CAs induced apoptosis by triggering ER stress. Because of their ease of synthesis and low GI50, these cephalostatin analogues represent promising anticancer drugs.