Oxysterols resulting from spontaneous or enzymatic oxidation of cholesterol are present in numerous foodstuffs and have been identified at increased levels in the plasma and the vascular walls of patients with cardiovascular diseases, especially in atherosclerotic lesions. Consequently, their role in lipid disorders is widely suspected, but they may also contribute to the development of important degenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, osteoporosis, age-related macular degeneration, and cataract. Since these pathologies can be associated with the presence of apoptotic cells, oxidative and inflammatory processes, and lipid disorders, the ability of oxysterols to trigger cell death, activate oxidation and inflammation, and modulate lipid homeostasis is being extensively studied. There are several important considerations regarding the physiological/pathophysiological functions and activities of the different oxysterols. It is therefore important to determine their biological activities and identify their signaling pathways, when they are used either in isolation or as mixtures. In these conditions, oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or no effects whatsoever. Moreover, with cytotoxic oxysterols, a substantial accumulation of polar lipids in cytoplasmic multilamellar structures was observed, demonstrating that cytotoxic oxysterols were phospholipidosis inducers. This basic knowledge on oxysterols contributes to a better understanding of the associated pathologies, so that new treatments and drugs can be designed.