Deciphering the molecular basis of leukocyte recruitment is critical to the understanding of inflammation. Here we show that the tetraspanin CD37 is a key player in leukocyte recruitment. Analyses of leukocyte-endothelial cell interactions by intravital microscopy revealed that responses induced by the neutrophil-attracting chemokine CXCL1 are reduced in CD37-/- mice. These mice show impairment of integrin-mediated processes including stable adhesion to the endothelium and transmigration from the vasculature, and CD37-/- neutrophils have a reduced capacity to adhere to the b2 integrin ligand ICAM-1 in vitro. Moreover, the absence of CD37 impairs the capacity of leukocytes to accurately follow a CXCL1 chemotactic gradient in the interstitium. To examine the mechanisms for these impairments, we used super-resolution microscopy of ICAM-1 adherent neutrophils to demonstrate that CD37 and b2 integrin segregate in both independent and overlapping clusters, with approximately 25% of b2 integrin clustered with CD37. Furthermore, ex vivo analyses revealed that the impaired functional capacity of b2 integrins in the absence of CD37 is associated with accelerated integrin internalization and reduced actin polymerization and cell spreading. These data provide evidence that the tetraspanin CD37 can cluster with b2 integrin in neutrophils and regulate integrin function by promoting outside-in signalling and cytoskeletal function downstream of integrin-mediated adhesion.