The cytotoxic activity of 7beta-hydroxycholesterol (7beta-OHC) was evaluated on human NCI-H460 lung cancer cells. 7beta-OHC decreased clonogenic survival of NCI-H460 in a dose dependent pattern. 7beta-OHC induced apoptosis in NCI-H460, with the characteristic features like increase in sub-G(1) hypodiploid (apoptotic) cells, and apoptotic body formation, as evidenced by flow cytometry and fluorescence microscope, respectively. Apoptosis was also associated with loss of mitochondrial transmembrane potential, and the activation of caspases 9 and 3. 7beta-OHC resulted in generation of reactive oxygen species (ROS) during apoptosis. On the whole, the results indicated that 7beta-OHC inhibited the proliferation of NCI-H460 cells through apoptosis via caspase activation.