The process of gamete fusion has been largely studied in the mouse and has revealed the crucial role of the tetraspanin CD9. By contrast, human gamete fusion remains largely unknown. We now show that an anti-alpha 6 integrin mAb (GoH3) strongly inhibited human sperm-egg fusion in human zona-free eggs. Furthermore, a mAb directed against CD151, a tetraspanin known to associate with alpha 6 beta1, partially inhibited sperm-egg fusion. By contrast, the addition of an anti-CD9 mAb to zona free eggs had no effect. The integrin alpha 6 beta1, CD151 and CD9 tetraspanins were evenly distributed on human zona-intact oocytes. On zona-free eggs, the integrin alpha 6 beta1 and tetraspanin CD151 patched and co-localized but the tetraspanin CD9 remained unchanged. CD9 mAb prevented alpha 6 beta1 integrin clustering and gamete fusion when added prior to, but not after, zona removal. Antibody-mediated aggregation of integrin alpha 6 beta1 yielded patches that were bigger and more heterogeneous in mouse oocytes lacking CD9. Moreover, a strong labelling of alpha 6 beta1 could be observed at the sperm entry point. Altogether, these data show that CD9 controls the redistribution of some membrane proteins including the alpha 6 beta1 integrin into clusters that may be necessary for gamete fusion.