α6β4 integrin is localized in a unique punctate distribution at the cell-substratum interface along the leading front of single, front-rear-polarized A549 cells. These puncta are interspersed between focal adhesions and lack association with the actin cytoskeleton. Knockdown of β4 integrin in A549 cells inhibits their directed migration, with knockdown cells exhibiting large focal adhesions and reduced actin dynamics. Despite these changes, the speed of knockdown cells is equivalent to control cells. Interestingly, in such cells, α6 integrin retains its punctate distribution. Moreover, in β4 integrin knockdown cells, we observe a loss of β1 integrin from focal adhesions and an enhanced association with α6 integrin. We confirmed the switch in the β integrin binding partner of α6 integrin in the knockdown cells by immunoprecipitation. We next investigated the role of β4 integrin in collective cell migration. Wounded monolayers of β4 integrin knockdown cells exhibit reduced collective migration compared with controls. When we forced expression of β4 integrin in the leader cells of wounded monolayers, collective migration was restored. Similarly, forced expression of β4 integrin in primary rat alveolar epithelial cells also promotes collective cell migration. In addition, we interrogated the pathway by which β4 integrin regulates A549 cell-directed migration. Constitutively active Ras-related C3 botulinum toxin substrate 1 rescues motility defects resulting from β4 integrin deficiency. Together, our results support the hypothesis that α6β4 integrin is a positive regulator of collective cell migration of A549 cells through influence on signal pathways in leader cells.