Pedaling is widely used for rehabilitation of locomotion because it induces similar muscle activity to that observed during locomotion. However, no study has examined the effects of pedaling exercise on intracortical inhibition. The aim of the present study was to investigate the effect of pedaling exercise on short-interval intracortical inhibition (SICI) in the cortical area controlling the tibialis anterior (TA) and soleus (SOL) muscles. Ten healthy adults participated in this study and were instructed to perform 7 min of active and passive pedaling. Paired pulse transcranial magnetic stimulation (TMS) was used to investigate the SICI. Using interstimulus intervals of 2–3 ms, the SICI of TA and SOL muscles was recorded at rest before and after the pedaling and repetitive ankle dorsiflexion tasks. SICI in both TA and SOL muscles decreased immediately after active pedaling. There were no significant changes in SICI after the passive pedaling and repetitive ankle dorsiflexion. A short-term, low-intensity active pedaling exercise decreases the intracortical inhibition of the leg area of the motor cortex. Our results suggest that pedaling has the potential to restore ambulation-inducing cortical reorganization among patients with central nervous system lesions.