Abstract: Objective To study the effects of N-acetylglucosamine on the activity of mushroom tyrosinase in vitro as well as on tyrosinase activity and melanogenesis in B16 melanoma cells. Methods Various concentrations （1.5 - 50 mmol/L） of N-acetylglucosamine were used to incubate with mushroom tyrosinase for 10 minutes following by the measurement of dopa oxidase activity of tyrosinase. B16 melanoma cells were cultured and treated with six concentrations （1.5 - 100 mmol/L） of N-acetylglucosamine for 72 hours; then, the cell proliferation of, melanogenesis and expression of tyrosinase protein and activity of tyrosinase in cultured B16 cells were measured by methyl thiazolyl tetrazolium （MTT） assay, sodium hydroxide （NaOH）-lysis method, immunocytochemical method and dopa oxidation assay, respectively. Results N-acetylglucosamine of 1.5, 3, 6.25, 25 and 50 mmol/L significantly inhibited the activity of mushroom tyrosinase compared with phosphate buffered saline （0.7380 ± 0.0254, 0.7293 ± 0.0382, 0.7247 ± 0.0389, 0.7233 ± 0.0186, 0.7043 ± 0.0166 vs 0.8183 ± 0.0326, P < 0.05 or 0.01）. After treatment with N-acetylglucosamine of 25, 50 and 100 mmol/L, a significant suppression was observed in cell proliferation （absorbance: 0.5410 ± 0.0496, 0.4480 ± 0.0246 and 0.1273 ± 0.0137 vs 0.6523 ± 0.0569） of and melanogenesis （absorbance at 460 nm: 0.0070 ± 0.0008, 0.0049 ± 0.0012 and 0.0015 ± 0.0014 vs 0.0096 ± 0.0014） in B16 melanoma cells. Also, decreased activity （absorbance at 460 nm: 0.1003 ± 0.0404 and 0.0130 ± 0.0053 vs 0.2283 ± 0.0691） and protein expression （13.2700 ± 0.9741 and 8.5667 ± 2.0345 vs 17.4703 ± 2.0583） of tyrosinase were noted in B16 cells treated with N-acetylglucosamine of 50 and 100 mmol/L. Conclusions These studies show that N-acetylglucosamine inhibits tyrosinase activity and melanogenesis in murine B16 melanoma cells. Hence, N-acetylglucosamine may serve as a skin lightening agent in the future.