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CD81 is a member of the tetraspanin family that has been described to have a key role in cell migration of tumor and immune cells. To unravel the mechanisms of CD81-regulated cell migration, we performed proteomic analyses that revealed an interaction of the tetraspanin C-terminal domain with the small GTPase Rac. Direct interaction was confirmed biochemically. Moreover, microscopy cross-correlation analysis demonstrated the in situ integration of both molecules into the same molecular complex. Pull-down experiments revealed that CD81-Rac interaction was direct and independent of Rac activation status. Knockdown of CD81 resulted in enhanced protrusion rate, altered focal adhesion formation, and decreased cell migration, correlating with increased active Rac. Reexpression of wild-type CD81, but not its truncated form lacking the C-terminal cytoplasmic domain, rescued these effects. The phenotype of CD81 knockdown cells was mimicked by treatment with a soluble peptide with the C-terminal sequence of the tetraspanin. Our data show that the interaction of Rac with the C-terminal cytoplasmic domain of CD81 is a novel regulatory mechanism of the GTPase activity turnover. Furthermore, they provide a novel mechanism for tetraspanin-dependent regulation of cell motility and open new avenues for tetraspanin-targeted reagents by the use of cell-permeable peptides. © 2013 Jo et al., PI080794 and PI11/01645 from the Instituto de Salud Carlos III; SAF2011-25834 and ERC AdG-2011; BIO2009-07990 from the Ministerio de Educación y Ciencia; CAM BIO/0194/2006 from Comunidad de Madrid, and RECAVA RD06/0014 from the Fondo de Investigaciones Sanitarias (Ministerio de Sanidad y Consumo, Instituto Salud Carlos III); National Institutes of Health grant GM23244 and the Cell Migration Consortium (U54 GM064346)