The tumor suppressor gene p16^INK4a inhibits the kinase activity of the cyclin-dependent kinase 4–6/cyclin D complexes and subsequent phosphorylation of critical substrates necessary for transit through the G1 phase of the cell cycle. Recent studies suggested that control of the G1/S boundary might not be the sole biological function of p16^INK4a. We hypothesized that p16^INK4a might influence hitherto unknown critical features of a malignant epithelial phenotype, such as anchorage dependence. Here we provide evidence that stable transfection of p16^INK4a restitutes apoptosis induction upon loss of anchorage (anoikis) in a variety of human cancer cells. Anoikis in p16^INK4a-transfected cells was evidenced by DNA fragmentation and poly(ADP-ribose) polymerase cleavage upon cultivation on polyhydroxyethylmethacrylate-coated dishes and was associated with suppression of anchorage-independent growth as well as complete loss of tumorigenicity. p16^INK4a-mediated anoikis was due to selective transcriptional upregulation of the α₅ integrin chain of the α₅β₁ fibronectin receptor as detected by FACS^® analysis, immunoprecipitation, Northern blotting, and nuclear run-on assays. Addition of soluble fibronectin and inhibitory α₅ antibodies to nonadherent cells completely abolished p16^INK4a-mediated anoikis, whereas laminin was ineffective. Furthermore, antisense-induced downregulation of the α₅ integrin chain in p16^INK4a-transfected cells restored resistance to anoikis. These data suggest a novel functional interference between a cell cycle–regulating tumor suppressor gene and membrane-bound integrins, thus regulating a hallmark feature of an epithelial transformed phenotype: susceptibility to anoikis.