Fibulin-5, an extracellular matrix glycoprotein expressed in elastin-rich tissues, regulates vascular cell behaviour and elastic fibre deposition. Recombinant full-length human fibulin-5 supported primary human aortic SMC (smooth-muscle cell) attachment through α5β1 and α4β1 integrins. Cells on fibulin-5 spread poorly and displayed prominent membrane ruffles but no stress fibres or focal adhesions, unlike cells on fibronectin that also binds these integrins. Cell migration and proliferation were significantly lower on fibulin-5 than on fibronectin. Treatment of cells on fibulin-5 with a β1 integrin-activating antibody induced stress fibres, increased attachment, migration and proliferation, and stimulated signalling of epidermal growth factor receptor and platelet-derived growth factor receptors α and β. Fibulin-5 also modulated fibronectin-mediated cell spreading and morphology. We have thus identified the β1 integrins on primary SMCs that fibulin-5 interacts with, and have shown that failure of fibulin-5 to activate these receptors limits cell spreading, migration and proliferation.