Pycnogenol is a natural plant extract from pine bark that contains compounds that have anti-oxidative, free-radical scavenging properties. In this work, utilizing cultured B16 melanoma cells (B16 cells), pycnogenol was investigated for its ability to inhibit tyrosinase activity and melanin biosynthesis. We also examined the anti-oxidative power of pycnogenol by measuring its suppressive effect against peroxynitrite (ONOO-), superoxide (.O2), nitric oxide (NO.), and hydroxyl radical (.OH)-scavenging activities using an electron spin resonance spectrometer. Results show that pycnogenol had a strong anti-tyrosinase activity and suppressed melanin biosynthesis. Further, our results showed that through its anti-oxidative properties, pycnogenol suppressed .O2) NO., ONOO-, and .OH in in vitro assays, and reactive species, ONOO-, .O2, and NO., while up-regulating the reduced glutathione/oxidized glutathione ratio in B16 cells. Based on the findings, we propose that pycnogenol exerts anti-melanogenic activity via its anti-oxidative actions.