CD151, one of the tetraspanins, forms a stable complex with integrin α3β1, the major laminin receptor on the cell surface. We found that 8C3, an anti-CD151 mAb obtained by screening for reactivity with integrin α3β1–CD151 complexes, was capable of dissociating CD151 from integrin α3β1, thereby allowing us to deplete CD151 from purified integrin α3β1–CD151 complexes. The CD151-free integrin α3β1 thus obtained showed a significant reduction in its ability to bind to laminin-10/11, a high-affinity ligand for integrin α3β1, with a concomitant reduction in its reactivity with mAb AG89, which recognizes activated β1-containing integrins. These results raised the possibility that the association of integrin α3β1 with CD151 potentiates the ligand-binding activity of the integrin through sustaining its activated conformation. In support of this possibility, the ligand-binding activity was restored when CD151-free integrin α3β1 was reassociated with purified CD151. 8C3-induced dissociation of CD151 from integrin α3β1 was also demonstrated on the surface of living cells by fluorescent resonance energy transfer imaging, accompanied by a concomitant reduction in the cell adhesion to laminin-10/11-coated substrates. CD151 knock-down by RNA interference also resulted in a reduction in the adhesive activity of the cells. Taken together, these results indicate that CD151 association modulates the ligand-binding activity of integrin α3β1 through stabilizing its activated conformation not only with purified proteins but also in a physiological context.