In transcranial magnetic stimulation (TMS) the regional electrical activity in the brain is influenced by a pulsed magnetic field. The rapidly changed magnetic field produces electrical currents that activate neurons. Repetitive TMS (rTMS) treatment can cause functional changes in the cortex. The present study clarified the effects of rTMS treatment on behavioral changes in rats, focusing on anxiety by using an elevated plus-maze (plus-maze) test. The effects of rTMS treatment on neurochemical changes during the plus-maze test were investigated by determining the extracellular levels of serotonin (5-HT) and dopamine (DA) in the prefrontal cortex by using in vivo microdialysis. Each rat received rTMS of the frontal brain for 3 days, during which 125 stimuli from five trains in a day were applied at 25 Hz for 1 s with 2-min intervals between trains. Three-day series of (chronic) rTMS treatment caused significant increases in the time spent in open arms and the number of entries into open arms of the plus-maze compared with non-treated and sham-treated rats, which were not observed in 1-day series of (acute) rTMS treatment. Chronic rTMS treatment suppressed the increases in 5-HT levels induced by the plus-maze test, but did not influence the elicited DA levels. These data suggest that chronic rTMS treatment of the frontal brain has anxiolytic effects in rats, which are related to the 5-HTergic neuronal system.