Galectin-3 is a family member of the carbohydrate-binding proteins widely expressed by many cell types and exhibits multiple cellular functions. We demonstrate that melanocytes express galectin-3, which is predominantly localized to the cell body peripherally along the Golgi zone. Downregulation of galectin-3 in human melanocytes using short hairpin RNA technology resulted in the reduction of both melanin synthesis and expression/activity of tyrosinase-related protein-1 (Tyrp-1). In the cell body, galectin-3 colocalizes with melanosome-destined cargo, specifically tyrosinase and Tyrp-1. We studied melanocytes cultured from patients with forms of Hermansky-Pudlak syndrome (HPS) containing defects in trafficking steps governed by biogenesis of lysosome-related organelle complex-2 (BLOC-2) (HPS-5), BLOC-3 (HPS-1), and adaptin-3 (HPS-2). We found that galectin-3 expression mimicked the defective expression of the tyrosinase cargo in dendrites of HPS-5 melanocytes, but it was not altered in HPS-1 or HPS-2 melanocytes. In addition, galectin-3 colocalized predominantly with the HPS-5 component of BLOC-2 in normal human melanocytes. These data indicate that galectin-3 is a regulatory component in melanin synthesis affecting the expression of Tyrp-1.