Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.