Tyrosinase is a key enzyme for melanin biosynthesis, and hyperpigmentation disorders are associated with abnormal accumulation of melanin pigments, which can be reduced by treatment with depigmenting agents. A methanol extract of Astragalus membranaceus showed inhibitory activity against mushroom tyrosinase. The active compound was purified from the methanol extract of A. membranaceus and, following several chromatographic methods, was identified as calycosin via spectroscopic analysis. The results showed that calycosin exhibited tyrosinase inhibitory activity with an IC₅₀ value of 38.4 μM. Moreover, calycosin showed a melanin biosynthesis inhibition zone in a culture plate of Streptomyces bikiniensis, which is commonly used as an indicator organism. Furthermore, calycosin dramatically reduced melanin synthesis of Melan-a cells without any apparent cytotoxicity and reduced expression of melanogenic enzyme, tyrosinase. These results suggest that calycosin may be an effective skin-lightening agent that regulates the expression of melanogenic enzymes.