Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via extracellular-regulated kinase (ERK). However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/minute. Phosphorylation of Src-Tyr⁴¹⁸, FAK-Tyr³⁹⁷-Tyr⁵⁷⁶-Tyr⁹²⁵ and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10µmol/L) or specific siRNA. Src inhibition also prevented strain-induced FAK phosphorylation at Tyr³⁹⁷ and Tyr⁵⁷⁶ but not FAK-Tyr⁹²⁵ or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of N-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F and Y397F-Y576F-Y577F did not prevent activation of ERK2 by cyclic strain but a FAK mutant at C-terminal Y925F prevented strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK-Tyr⁹²⁵ phosphorylation under static conditions, it nevertheless exhibited increased FAK-Tyr⁹²⁵ phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-Tyr⁹²⁵-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting.