As an essential lipid, cholesterol is of great value in keeping cell homeostasis, being the precursor of bile acid and steroid hormones, and stabilizing membrane lipid rafts. As a kind of cholesterol metabolite produced by enzymatic or radical process, oxysterols have drawn much attention in the last decades. Among which, the role of 25-hydroxycholesterol (25-HC) in cholesterol and bile acid metabolism, antivirus process, and inflammatory response has been largely disclosed. This review is aimed at revealing these functions and underlying mechanisms of 25-HC.