Retinal pigment epithelial (RPE) cells are among the most active phagocytes in the body. Every morning, circadian shedding of outer segment fragments by photoreceptor cells activates a synchronized phagocytic response by RPE cells that is critical for vision. RPE cells require alpha v beta5 integrin receptors for particle binding that triggers engulfment. Here, we show that tetraspanins CD81 and CD9 reside in a complex specifically with alpha v beta5 integrin but not the engulfment receptors Mer tyrosine kinase and CD36 at the apical, phagocytic surface of RPE cells. Function blocking and RNA silencing of CD81 but not of CD9 specifically diminish particle binding. CD81 but not CD9 overexpression is sufficient to increase particle binding and surface levels of alpha v beta5 integrin. Wild-type and mutant RPE cells defective in particle engulfment equally reduce and increase particle binding in response to CD81 inhibition and CD81 overexpression, respectively. By striking contrast, neither CD81 inhibition nor CD81 overexpression has any effect on particle binding by RPE lacking alpha v beta5 integrin. These results identify a novel and important role for CD81 in phagocytosis. CD81 does not function as a binding receptor by itself but promotes outer segment particle binding through functional interaction specifically with alpha v beta5 integrin.