Oxycholesterols (OS) are formed from cholesterol or its immediate precursors by enzymatic or free radical action in vivo, or they may be derived from food. OS exhibit a wide spectrum of biological activities. In OS cytotoxicity, several mechanisms seem to be involved: e.g. inhibition of HMG-CoA reductase activity, antiproliferative action, apoptosis induction, replacement of cholesterol by OS in membranes followed by changes in cellular membrane structure and functionality, and immune system functions alteration. Furthermore, OS may be mutagenic and carcinogenic and may serve as intracellular signaling or regulatory molecules. Here we review OS cellular activities with special attention to the cytotoxic action in vivo and in vitro using experimental models.