Voluntary exercise has been associated with reduced anxiety across several animal models. Manipulation of central 5-HT can alter anxiety-like behaviors and administration of the 5-HT agonist metachlorophenylpiperazine (mCPP) increases anxiety in rodents and humans. To examine whether the anxiolytic effect of exercise is associated with an alteration in 5-HT systems, we examined the anxiogenic effect of mCPP in exercising and nonexercising mice. C57BL/6J mice were given 2 weeks of free access to either a functioning or nonfunctioning running wheel. Mice were then tested for acoustic startle following systemic injection of either 0, 0.1, 0.3, or 1 mg/kg of mCPP. Consistent with its anxiogenic properties, mCPP produced a dose-dependent increase in acoustic startle in nonexercising mice. However, this anxiogenic effect was blunted in exercising mice. These findings suggest that exercise may help to reduce anxiety by altering 5-HT systems, perhaps by down-regulating postsynaptic 5HT 2B/2C receptors.