Profilin was first identified as an actin monomer binding protein; however, recent reports indicate its involvement in actin polymerization. To date, there is no direct evidence of a functional role in vivo for profilin in actin cytoskeletal reorganization. Here, we prepared a profilin mutant (H119E) defective in actin binding, but retaining the ability to bind to other proteins. This mutant profilin I suppresses actin polymerization in microspike formation induced by N‐WASP, the essential factor in microspike formation. Profilin associates both in vivo and in vitro with N‐WASP at proline‐rich sites different from those to which Ash/Grb2 binds. This association between profilin and N‐WASP is required for N‐WASP‐induced efficient microspike elongation. Moreover, we succeeded in reconstituting microspike formation in permeabilized cells using profilin I combined with N‐WASP and its regulator, Cdc42. These findings provide the first evidence that profilin is a key molecule linking a signaling network to rapid actin polymerization in microspike formation.