CD81 (TAPA-1) is a member of the widely expressed and evolutionary conserved tetraspanin family that forms complexes with a variety of other cell surface receptors and facilitates hepatitis C virus entry. Here, we show that CD81 is specifically required for the formation of lamellipodia in migrating dendritic cells (DCs). Mouse CD81(-/-) DCs, or murine and human CD81 RNA interference knockdown DCs lacked the ability to form actin protrusions, thereby impairing their motility dramatically. Moreover, we observed a selective loss of Rac1 activity in the absence of CD81, the latter of which is exclusively required for integrin-dependent migration on 2-dimensional substrates. Neither integrin affinity for substrate nor the size of basal integrin clusters was affected by CD81 deficiency in adherent DCs. However, the use of total internal reflection fluorescence microscopy revealed an accumulation of integrin clusters above the basal layer in CD81 knockdown cells. Furthermore, β1- or β2-integrins, actin, and Rac are strongly colocalized at the leading edge of DCs, but the very fronts of these cells protrude CD81-containing membranes that project outward from the actin-integrin area. Taken together, these data suggest a thus far unappreciated role for CD81 in the mobilization of preformed integrin clusters into the leading edge of migratory DCs on 2-dimensional surfaces.