ABSTRACT. The melanin granules are synthesized in melanocytes in the epidermal basal layer and the hair matrix. For the effective passage of melanin granules to the adjacent keratinocytes, melanocytes utilize unique cytoplasmic delivery system in which cytoskeletal network is prominently involved. Here, we show that the t-SNARE protein syntaxin3, a member of a family of key mediators of the cytoplasmic vesicle fusion and potent modulators of cytoskeletal dynamics, dramatically affects melanocyte cell behavior. Although plasmalemmal syntaxin3 has been detected also on the melanosomes of normal human melanocytes, we noticed that mouse melanoma B16 cells had completely lost endogenous syntaxin3. In response to the forcible expression of syntaxin3, B16 cells formed well-developed dendritic filopodia and accumulated melanin granules in the cytoplasm. We found that exogenous syntaxin3 was not expressed at the plasma membrane, but rather, localized with non-fibrous F-actin and melanin-packed melanosomes in the cytoplasm, by which the assembly/ polymerization of actin was dramatically impacted and the melanosome secretion was severely suppressed. The syntaxin3-triggered phenotypic changes were also induced by a syntaxin3 mutant lacking SNARE and transmembrane domains, and they were completely reverted by the subsequent knockdown of exogenous syntaxin3. This t-SNARE protein may act as a regulator of the actin dynamics, rather than a direct vesicle fusion mediator, to determine the fundamental properties of melanocytes.