Tetraspanins are highly conserved membrane proteins that function as membrane-organizers and control fundamental cell functions, including adhesion, migration and proliferation. The tetraspanin CD9 specifically associates with different adhesion receptors of the immunoglobulin and integrin families in tetraspanin-enriched microdomains (TEMs). Through these interactions, CD9 exerts important regulatory effects on the function of associated adhesion molecules. Our group has reported that CD9 associates directly with the metalloproteinase ADAM17 on the surface of different types of cells, exerting an inhibitory effect on ADAM17 sheddase activity against a variety of its substrates. Interactions of integrin α5β1 with ADAM17 may occur among molecules expressed on the same cell (cis) or on different cells (trans), with the latter reported to support cell-cell adhesion events. Interestingly, the interaction between integrin α5β1 and the disintegrin domain of ADAM17 has been shown to cause the inhibition of both the adhesive capacity of the integrin (i.e. its ability to bind its ligands) as well as of ADAM17 metalloproteinase activity due to steric hindrance leading to decreased accessibility of its catalytic site for the substrate. In contrast, stimuli that promote the dissociation of the α5β1-ADAM17 complex induce the activation of ADAM17 sheddase activity and increase integrin-mediated cell adhesion. Interestingly, the expression of CD9 on the cell surface abrogates integrin α5β1-mediated cell adhesion to its ligands fibronectin and ADAM17-Fc. In situ proximity ligation assays (PLA) and biochemical experiments based on co-immunoprecipitation collectively revealed that the mechanism by which CD9 inhibits α5β1-mediated cell adhesion involves the reinforcement of cis interactions between ADAM17 and α5β1 on the cell surface, which takes place without alteration in α5β1 integrin affinity but is evidenced by changes in the organization of integrin molecules at the plasma membrane.