Melanogenic enzymes are transported by vesicular/membrane trafficking to immature melanosomes in melanocytes where they catalyze the synthesis of melanin pigments. Although several factors involved in melanogenic enzyme trafficking have been identified in the past decade, involvement of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, which generally mediate membrane fusion, on melanosomes in the process of melanogenic enzyme trafficking has never been investigated. In this study we identified syntaxin-3, which was originally described as a target SNARE protein at the plasma membrane, as a melanosome-resident protein and investigated whether syntaxin-3 is involved in the trafficking of the melanogenic enzyme Tyrp1 (tyrosinase-related protein 1) in mouse melanocytes. The results showed that knockdown of endogenous syntaxin-3 protein in melanocytes caused a dramatic reduction in Tyrp1 signals, especially from peripheral melanosomes, presumably as a result of lysosomal degradation of Tyrp1. They also showed that syntaxin-3 interacts with another target SNARE SNAP23 (synaptosome-associated protein of 23 kDa) and with vesicle SNARE VAMP7 (vesicle-associated membrane protein 7), which has been shown to be localized at Tyrp1-containing vesicles/organelles. These findings suggested that the SNARE machinery composed of VAMP7 on Tyrp1-containing vesicles and syntaxin-3 and SNAP23 on melanosomes regulates Tyrp1 trafficking to the melanosome in melanocytes.