Disruption of intercellular adhesions, increased abundance of α5β1 integrin, and activation of protein kinase Cɛ (PKCɛ) correlate with invasion and unfavorable prognosis in lung cancer. However, it remains elusive how these distinct factors contribute to the invasive behavior of cancer cells. Persistent cell motility requires the formation of stable lamellae at the leading edge of a migrating cell. Here, we report that the tight junction protein zonula occludens-1 (ZO-1) preferentially interacts with α5β1 integrin at the lamellae of migrating cells. Disruption of ZO-1 binding to an internal PDZ-binding motif in the α5 cytoplasmic tail prevented the polarized localization of ZO-1 and α5 at the leading edge. Furthermore, silencing of α5 integrin inhibited migration and invasion of lung cancer cells, and silencing of ZO-1 resulted in increased Rac activity and reduced directional cell motility. The formation of the α5–ZO-1 complex was dependent on PKCɛ: Phosphorylation of ZO-1 at serine-168 regulated the subcellular localization of ZO-1 and thus controlled its association with α5 integrin. In conclusion, PKCɛ activation drives the formation of a spatially restricted, promigratory α5–ZO-1 complex at the leading edge of lung cancer cells.