In previous studies, 4-hydroxy-5-methyl-3[2H]-furanone (HMF) was shown to have potent antioxidative and antimelanogenic effects, suggesting its potential use as a depigmenting agent. The present study investigated its mechanism of action on murine melanoma B16F10 cells stimulated by theophylline, an activator of the cyclic AMP/protein kinase A signaling leading to tyrosinase gene expression. When the cells were stimulated with theophylline, there were dose-dependent increases in cellular tyrosinase protein content and melanin formation, as expected. HMF inhibited the theophylline-stimulated melanin formation as effectively as arbutin, one of the most widely used depigmenting agents in cosmetics. HMF appeared to reduce tyrosinase mRNA and protein content in the cells stimulated by theophylline, indicating it inhibited tyrosinase gene expression. HMF also effectively inhibited tyrosinase-catalyzed melanin formation from dihydroxyphenylalanine in the cells as well as in vitro. Therefore, the antimelanogenic effects of HMF were best explained by a dual mechanism inhibiting tyrosinase gene expression and the enzyme activity of pre-existing tyrosinase.