Sphingomyelin (SM) is a major component of the plasma membrane in mammalian cells, and a major component of plasma lipids, which are packaged in lipoprotein particles of varying densities. SM can be synthesized de novo from serine and palmitoyl-CoA, catalyzed by the rate-limiting enzyme serine palmitoyltransferase. The antibiotic myriocin potently inhibits serine palmitoyltransferase and decreases the accumulation of SM and other sphingolipids. Sphingolipid depletion leads to reduced dietary cholesterol absorption and increased reverse cholesterol transport. Recently, we demonstrated that SM depletion increases cholesterol efflux in both ABCA1-dependent and -independent manners, which was accompanied by increased cell surface phosphatidylserine and the reduced inward translocation of phosphatidylserine. This review will discuss SM's role in maintaining plasma membrane asymmetry and lipid raft domains, HDL metabolism, reverse cholesterol transport, and how SM depletion alters plasma membrane structure and function, leading to modulation of cholesterol extractability by lipoproteins and other acceptors.