Aberrant adhesion signaling pathways in cancer cells underlie their deadly invasive capabilities. The adhesion-related PDZ adapter protein mda-9/syntenin is a positive regulator of cancer cell progression in breast cancer, melanoma, and other human cancers. In this study, we report that mda-9/syntenin mediates adhesion-mediated activation of protein kinase Calpha (PKCalpha) and focal adhesion kinase (FAK) by fibronectin (FN) in human breast cancer and melanoma cells. FN rapidly stimulated the expression of mda-9/syntenin and the activation of PKCalpha prior to activation of FAK. Inhibiting PKCalpha suppressed basal or FN-induced expression of mda-9/syntenin, as well as cell migration and invasion toward FN stimulated by mda-9/syntenin. Several lines of evidence suggested that activation of PKCalpha and expression of mda-9/syntenin were interdependent. First, mda-9/syntenin inhibition suppressed basal or FN-induced phosphorylation of PKCalpha at Thr(638/641), whereas PKCalpha inhibition suppressed basal or FN-induced expression of mda-9/syntenin. Second, inhibiting either mda-9/syntenin or PKCalpha suppressed FN-induced formation of integrin-beta(1)/FAK/c-Src signaling complexes. Third, inhibiting either mda-9/syntenin or PKCalpha suppressed FN-induced phosphorylation of FAK Tyr(397) and c-Src Tyr(416) and the induction of downstream effector signals to p38 and mitogen-activated protein kinase, Cdc42, and NF-kappaB. In summary, our findings offer evidence that mda-9/syntenin acts as a molecular adaptor linking PKCalpha and FAK activation in a pathway of FN adhesion by human breast cancer and melanoma cells.