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Nicotine withdrawal symptoms comprise insomnia, depression, anxiety, attention disorders, and increased craving. We evaluated the amelio-rating effect of treadmill exercise on nicotine withdrawal symptoms. The rats in the nicotine withdrawal groups received subcutaneous in-jection with 6-mg/kg nicotine hydrogen tartrate salt for 17 days. And then, the injection of nicotine hydrogen tartrate salt was stopped next for 2 weeks. The rats in the exercise groups performed treadmill run-ning once a day, 5 days per week, for 31 days. In the present results, activity was decreased and anxiety-like behavior was observed in the nicotine withdrawal rats. Treadmill running increased activity and ame-liorated anxiety-like behavior in the nicotine-withdrawal rats. Expres-sions of tryptophan hydroxylase (TPH) and 5-hydroxytryptamine (5-HT) in the dorsal raphe were decreased in the nicotine withdrawal rats, in contrast, treadmill running increased TPH and 5-HT expressions. Im-paired short-term memory and deteriorated spatial learning ability were observed in the nicotine withdrawal rats, in contrast, treadmill running ameliorated impairment of short-term memory and spatial learning abil-ity. Expressions of brain-derived neurotrophic factor and tyrosine kinase B (TrkB) were decreased in the nicotine withdrawal rats, in contrast, treadmill running increased brain-derived neurotrophic factor and TrkB expressions. The numbers of the dou¬blecortin (DCX)-positive cells and 5-bromo-2´-deoxyuridine (BrdU)-positive cells in the dentate gyrus were suppressed in the nicotine withdrawal rats, in contrast, treadmill run-ning enhanced the numbers of DCX-positive cells and BrdU-positive cells. The present study demonstrate that treadmill exercise ameliorat-ed nicotine withdrawal-induced anxiety, depression, and memory im-pairment.