Engagement of α5β1-integrin by fibronectin (FN) acutely enhances Cav1.2 channel (CaL) current in rat arteriolar smooth muscle and human embryonic kidney cells (HEK293-T) expressing CaL. Using coimmunoprecipitation strategies, we show that coassociation of CaL with α5- or β1-integrin in HEK293-T cells is specific and depends on cell adhesion to FN. In rat arteriolar smooth muscle, coassociations between CaL and α5β1-integrin and between CaL and phosphorylated c-Src are also revealed and enhanced by FN treatment. Using site-directed mutagenesis of CaL heterologously expressed in HEK293-T cells, we identified two regions of CaL required for these interactions: 1) COOH-terminal residues Ser1901 and Tyr2122, known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively; and 2) two proline-rich domains (PRDs) near the middle of the COOH terminus. Immunofluorescence confocal imaging revealed a moderate degree of wild-type CaL colocalization with β1-integrin on the plasma membrane. Collectively, our results strongly suggest that 1) upon ligation by FN, CaL associates with α5β1-integrin in a macromolecular complex including PKA, c-Src, and potentially other protein kinases; 2) phosphorylation of CaL at Y2122 and/or S1901 is required for association of CaL with α5β1-integrin; and 3) c-Src, via binding to PRDs that reside in the II–III linker region and/or the COOH terminus of CaL, mediates current potentiation following α5β1-integrin engagement. These findings provide new evidence for how interactions between α5β1-integrin and FN can modulate CaL entry and consequently alter the physiological function of multiple types of excitable cells.