<p>Tetraspanin CD82/KAI1 inhibits cell movement and metastasis of malignant tumors, and reduced and lost expressions of CD82 predict worse outcomes of patients with malignant tumors. Here we found that CD82 inhibits both solitary and collective movement of tumor cells. The CD82 YVAA mutation, which affects CD82 trafficking, selectively abrogates CD82-mediated inhibition of collective migration. Cilengitide, at the concentration that specifically inhibits integrin αVβ3, also selectively blocks collective movement, underscoring a promotive role of integrin αVβ3 in this mode of cell motility. In contrast, integrin αVβ5 appears non-essential for collective migration, and both αVβ3 and αVβ5 are dispensable for solitary movement on fibronectin, highlighting distinct functions of different integrins in different modes of tumor cell movement. CD82 interacts with αVβ3 and αVβ5 integrins and downregulates their protein levels, while CD82 YVAA mutation relinquishes this downregulation without disrupting CD82 interactions with these integrins. Mechanistically, CD82, but not the YVAA mutant, considerably reduces digitation junction—the structure where integrin αVβ3 localizes—and likely directs integrin αVβ3 for lysosomal degradation, thereby lowering its level and suppressing collective migration. Thus, our study reveals that i) integrin αVβ3 promotes collective movement of tumor cells, ii) CD82 counteracts this by diminishing integrin αVβ3 and its presence in microextrusions, and iii) digitation junction likely participates in collective cell movement. Our study further demonstrates that endolysosomal trafficking of CD82 and integrin αVβ3 is needed for their collective movement-regulatory activities and that coupling of metastasis suppressor CD82/KAI1 with different partners regulates different modes of cell movement.</p>