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The coat color of mammals is primarily determined by the type, quantity, and distribution of melanin in the skin and hair. As an endogenous gas molecule, nitric oxide (NO) regulates tyrosinase production by modulating the cGMP-dependent protein kinase (PKG) pathway, which enhances melanin synthesis. However, some interrelationships have not been fully elucidated. In the present study, mouse melanocytes co-cultured with mouse keratinocytes in vitro, or as monocultures, were used as research models. The results indicated that ultraviolet B irradiation increased nitric oxide synthase (NOS) activity and NO production, and increased PKG, p21-activated kinase 4 (PAK4), and microphthalmia-associated transcription factor (MITF) levels, as well as tyrosinase (TYR), tyrosinase-related protein 1 and 2 expression, and melanin synthesis. During PKG inhibition, the expression of NO-regulated PAK4 and MITF was decreased. Pigment production was also affected, but remained higher than that in the control and NO inhibitor groups. These findings suggest that ultraviolet light regulates melanin production by activating the NO/cGMP/PKG pathway, which mediates the expression of PAK4, affecting melanin synthesis. On this basis, further elucidation of this regulatory network may improve our understanding of patterns of animal hair color formation.