Physical forces including pressure, strain, and shear can be converted into intracellular signals that regulate diverse aspects of cell biology. Exposure to increased extracellular pressure stimulates colon cancer cell adhesion by a 1-integrin-dependent mechanism that requires an intact cytoskeleton and activation of FAK and Src. -Actinin facilitates focal adhesion formation and physically links integrin-associated focal adhesion complexes with the cytoskeleton. We therefore hypothesized that -actinin may be necessary for the mechanical response pathway that mediates pressure-stimulated cell adhesion. We reduced -actinin-1 and -actinin-4 expression with isoform-specific siRNA. Silencing of -actinin-1, but not -actinin-4, blocked pressure-stimulated cell adhesion in human SW620, HT-29, and Caco-2 colon cancer cell lines. Cell exposure to increased extracellular pressure stimulated -actinin-1 tyrosine phosphorylation, -actinin-1 interaction with FAK and/or Src, and enhanced FAK phosphorylation at residues Y397 and Y576. The requirement for -actinin-1 phosphorylation in the pressure response was investigated by expressing the -actinin-1 tyrosine phosphorylation mutant Y12F in the colon cancer cells. Expression of Y12F blocked pressure-mediated adhesion, and inhibited the pressure-induced association of -actinin-1 with FAK and Src, and FAK activation. Furthermore, siRNA-mediated reduction of -actinin-1 eliminated the pressure-induced association of -actinin-1 and Src with 1-integrin receptor, and FAK-Src complex formation. These results suggest that -actinin-1 phosphorylation at tyrosine 12 plays a crucial role in pressure-activated cell adhesion and mechanotransduction by facilitating Src recruitment to 1-integrin, and consequently the association of FAK with Src, to enhance FAK phosphorylation.