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Melatonin, an indolamine produced and secreted predominately by the pineal gland, exhibits a variety of physiological functions, possesses antioxidant and antitumor properties. In this study, we have shown that pharmacologic concentration (1 mm) of melatonin significantly reduced cell migration and invasion of T98G and U251 glioma cells after 24-hr treatment and inhibited expression of matrix metalloproteinase 2 (MMP 2) and MMP 9. The melatonin inhibition of cell migration and invasion was associated with its reduction of intracellular basal free radical generation. Melatonin at pharmacologic concentration also inhibited the constitutive activation of the reactive oxygen species downstream transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Furthermore, pyrrolidine dithiocarbamate, a NF-κB-specific inhibitor, at 10 μm displayed anti-migration and invasion effects and inhibition of MMP 2 and MMP 9 expression resembling that of melatonin. Taken together, it is concluded that inhibition of migration and invasion of glioma cells by melatonin is associated with the latter in its inhibition of oxidative stress pathway. This suggests a potential therapeutic application of melatonin in the treatment of glioma.