Rhamnetin is a naturally occurring polyphenolic compound. In this report, experimental evidence is presented on the suppression of melanogenesis by rhamnetin using B16 murine melanoma cells (B16 cells). To document the underlying anti-melanogenic action of rhamnetin, several key biochemical mediators were quantified: superoxide (O₂^•−), nitric oxide (·NO) and peroxynitrite (ONOO⁻) in vitro , and total reactive species (RS) generation, O₂^•−, ·NO and ONOO⁻, reduced glutathione (GSH)/GSH-to-oxidized glutathione (GSSG) ratio, prostaglandin E₂ (PGE₂) and thromboxane B₂ (TXB₂) in B16 cells. Results revealed that rhamnetin inhibited murine tyrosinase activity, suppressed melanin content and oxidative stress, reducing O₂^•−,·NO and ONOO⁻ in vitro and total RS generation, O₂^•−, ·NO and ONOO⁻ in B16 cells, while maintaining a well-balanced GSH/GSSG ratio in B16 cells. Results further revealed that rhamnetin suppressed key pro-inflammatory mediators such as PGE₂ and TXB₂. Thus, these results strongly indicate that rhamnetin has powerful anti-melanogenic properties through its anti-oxidative and anti-inflammatory actions.