Oxysterol-binding protein (OSBP)-related protein 3 (ORP3) is highly expressed in epithelial, neuronal and hematopoietic cells, as well as in certain forms of cancer. We assessed the function of ORP3 in HEK293 cells and in human macrophages. We show that ORP3 interacts with R-Ras, a small GTPase regulating cell adhesion, spreading and migration. Gene silencing of ORP3 in HEK293 cells results in altered organization of the actin cytoskeleton, impaired cell-cell adhesion, enhanced cell spreading and an increase of β1 integrin activity–effects similar to those of constitutively active R-Ras(38V). Overexpression of ORP3 leads to formation of polarized cell-surface protrusions, impaired cell spreading and decreased β1 integrin activity. In primary macrophages, overexpression of ORP3 leads to the disappearance of podosomal structures and decreased phagocytotic uptake of latex beads, consistent with a role in actin regulation. ORP3 is phosphorylated when cells lose adhesive contacts, suggesting that it is subject to regulation by outside-in signals mediated by adhesion receptors. The present findings demonstrate a new function of ORP3 as part of the machinery that controls the actin cytoskeleton, cell polarity and cell adhesion.