Human StarD5 belongs to the StarD4 subfamily of START (for steroidogenic acute regulatory lipid transfer) domain proteins. We previously reported that StarD5 is located in the cytosolic fraction of human liver and binds cholesterol and 25-hydroxycholesterol. After overexpression of the gene encoding StarD5 in primary rat hepatocytes, free cholesterol accumulated in intracellular membranes. These findings suggested StarD5 to be a directional cytosolic sterol transporter. The objective of this study was to determine the localization of StarD5 in human liver. Western blot analysis confirmed StarD5's presence in the liver but not in human hepatocytes. Immunohistochemistry studies showed StarD5 localized within sinusoidal lining cells in the human liver and colocalized with CD68, a marker for Kupffer cells. Western blot analyses identified the presence of StarD5 in monocytes and macrophages as well as mast cells, basophils, and promyelocytic cells, but not in human hepatocytes, endothelial cells, fibroblasts, osteocytes, astrocytes, or brain tissue. Cell fractionation and immunocytochemistry studies on THP-1 macrophages localized StarD5 to the cytosol and supported an association with the Golgi. The presence of this cholesterol/25-hydroxycholesterol-binding protein in cells related to inflammatory processes provides new clues to the role of this protein in free sterol transport in the cells and in lipid-mediated atherogenesis.