Nicotine is an alkaloid found in tobacco leaves. Smoking prevention has been a neglected issue in psychiatry; nicotine intake in conjunction with the administration of the mood stabilizer, lithium carbonate (Li2CO3), may negatively affect brain cells. The present study investigated the combined effects of nicotine and its metabolite, cotinine, and Li2CO3 compared to acetylcholine and dopamine in U-251MG human glioblastoma cells. Cell proliferation was found to be decreased by nicotine and to be further suppressed following treatment with Li2CO3, accompanied by mitotic catastrophe and increased levels of superoxide anion radicals. By contrast, cotinine did not exert such detrimental effects. It was also found that acetylcholine did not suppress cell proliferation, whereas dopamine in conjunction with Li2CO3 decreased cell proliferation in a concentration-dependent manner. The nicotine-induced cell growth inhibition was restored by mecamylamine, a non-competitive antagonist of nicotinic acetylcholine receptors. On the whole, the findings of the present study suggest that nicotine combined with Li2CO3 leads to the suppression of the proliferation of human glioblastoma cells accompanied by mitotic catastrophe and superoxide anion radical generation. These findings may provide further cellular biological insight into the risks associated with smoking under Li2CO3 administration.