Melanoregulin (Mreg), a dilute suppressor gene product, has been implicated in the regulation of melanosome transport in mammalian epidermal melanocytes, because Mreg deficiency was found to restore peripheral melanosome distribution from perinuclear melanosome aggregation in Rab27A-deficient melanocytes. However, the function of Mreg in melanosome transport has remained unknown. Here we show that Mreg regulates microtubule-dependent retrograde melanosome transport through the dynein-dynactin motor complex. Mreg interacted with the C-terminal domain of RILP (Rab interacting lysosomal protein) and formed a complex with RILP and p150Glued, a component of the dynein-dynactin motor complex, in cultured cells. Overexpression of Mreg, RILP, or both in normal melanocytes induced perinuclear melanosome aggregation, whereas knockdown of Mreg or functional disruption of the dynein-dynactin motor complex restored peripheral melanosome distribution in Rab27A-deficient melanocytes. These findings reveal a novel mechanism by which the dynein-dynactin motor complex recognizes Mreg on mature melanosomes through interaction with RILP and is involved in their centripetal movement.