Abstract: Objective To investigate the role of autophagy in the regulatory effect of Shufeng Huoxue Fumula (SFHXF) on the proliferation and melanin metabolism in cultured murine B16 melanoma cells. Methods B16 cells were treated with solutions containing 0.12, 0.25, 0.49, 0.98, or 1.96 mg/mL SFHXF preparations, rapamycin (an autophagy inducer), or rapamycin+SFHXF. The changes in the proliferation of B16 cells were assessed using MTT assay, and tyrosinase activity and melanin content in the cells were determined. The expressions of autophagy- related proteins P62, p-mTOR, LC3B, and beclin 1 in the cells were detected using Western blotting. Result Compared with the blank control cells, treatments with SFHXF both in the presence and in the absence of rapamycin concentration-dependently inhibited the cell proliferation (P<0.05) and obviously increased tyrosinase activity and melanogenesis in B16 cells (P<0.05); 0.98 mg/mL SFHLF, rapamycin+0.98 mg/mL SFHXF, and 50 nmol/L rapamycin all significantly up-regulated the expressions of LC3B-II and beclin 1 and down-regulated the expressions of P62 and p-mTOR in the cells. Conclusion SFHXF can regulate melanin metabolism and enhance tyrosinase activity and melanogenesis through the autophagy pathway to inhibit the proliferation of B16 cells in vitro.