The intestinal epithelium is subjected to various types of mechanical stress. In this study, we investigated the impact of cyclic stretch on tight junction and adherens junction integrity in Caco-2 cell monolayers. Stretch for 2 hours resulted in dramatic modulation of tight junction protein distribution from linear organization into wavy structure. Continuation of cyclic stretch for 6 hours led to redistribution of tight junction proteins from the intercellular junctions into intracellular compartment. Disruption of tight junctions was associated with redistribution of adherens junction proteins, E-cadherin and β-catenin, and dissociation of the actin cytoskeleton at the actomyosin belt. Stretch activates JNK2, c-Src and MLCK. Inhibition of JNK, Src kinase or MLCK activity and knockdown of JNK2 or c-Src attenuated stretch-induced disruption of tight junctions, adherens junctions and actin cytoskeleton. Stretch increased tyrosine phosphorylation of occludin, ZO-1, E-cadherin and β-catenin. Inhibition of JNK or Src kinase attenuated stretch-induced occludin phosphorylation. Immunofluorescence localization indicated that phospho-MLC co-localizes with the vesicle-like actin structure at the actomyosin belt in stretched cells. On the other hand, phospho-c-Src co-localizes with the actin at the apical region of cells. This study demonstrates that cyclic stretch disrupts tight junctions and adherens junctions by a JNK2, c-Src and MLCK-dependent mechanism.