41 integrin plays a pivotal role in cell migration in vivo. This integrin has been shown to regulate front-back polarity of migrating cells via localized inhibition of 4 integrin/paxillin binding by phosphorylation at the 4 integrin cytoplasmic tail. Here we demonstrate that 41 regulates directionally persistent cell migration via a more complex mechanism in which 4 phosphorylation and paxillin-binding act via both cooperative and independent pathways. We show that, in response to shear flow, 41 binding to the CS-1 region of fibronectin was necessary and sufficient to promote directionally persistent cell migration when this integrin was ectopically expressed in CHO cells. Under shear flow, the 41-expressing cells formed a fan shape with broad lamellipodia at the front and retracted trailing edges at the back. This 'fanning' activity was enhanced by disrupting paxillin-binding alone and inhibited by disrupting phosphorylation alone or together with disrupting paxillin-binding. Notably, the phosphorylation-disrupting mutation and the double mutation resulted in the formation of long trailing tails, suggesting that 4 phosphorylation is required for trailing edge retraction/detachment independent of paxillin-binding. Furthermore, the stable polarity and directional persistence of shear flow-stimulated cells were perturbed by the double mutation but not the single mutations alone, indicating that paxillin-binding and 4 phosphorylation can facilitate directionally persistent cell migration in an independent and compensatory manner. These findings provide a new insight into the mechanism by which integrins regulate directionally persistent cell migration.