Abstract: Background and Objective: Thymoquinone (TQ) is one of the most potent constituents in Nigella Sativa and has many medicinal effects. The Rural Development Administration of Korea that is using genetic engineering to combine the properties of resveratrol and rice develops Iksan526. The present study was aimed to investigate the effects of thymoquinone (TQ) and Iksan526 callus extract (IS526) on A375 and B16F10 melanoma cell lines. Materials and Methods: A375 and B16F10 cells, cultured in vitro, were treated with TQ or IS526 and their effects on cell proliferation, tyrosinase activity, Reactive Oxygen Species generation (ROS), and expression levels of Cyclooxygenase (COX)-2, phosphorylated p38, and phosphorylated extracellular signal-regulated kinase (pERK) were evaluated with the MTT assay, tyrosinase activity assay, ROS assay, and western blot analysis, respectively. Results: In comparison with cells treated with either TQ or IS526, those treated with both TQ and IS526 showed a significant decrease in cell proliferation. TQ-induced cyclooxygenase-2 (COX-2) was inhibited by IS526 and TQ inhibited IS526-induced ROS production via MAPKs pathway in A375. Also, co-treatment with TQ and IS562 decreased tyrosinase expression via MAPKs pathway in A375. Combination with TQ and IS526 suppressed COX-2 and tyrosinase expression via the p38 kinase pathway in B16F10 cells. Conclusion: Taken together, results demonstrated that combination with TQ and IS526 can suppress cell growth and regulates COX-2, tyrosinase and ROS in melanoma cells, indicating its application potential for melanoma therapy.