We previously reported that even low-intensity, short-duration acute aerobic exercise decreases arterial stiffness. We aimed to test the hypothesis that the exercise-induced decrease in arterial stiffness is caused by the increased production of nitric oxide (NO) in vascular endothelium with exercise. Nine healthy men (age: 22~28 yr) performed a 5-min single-leg cycling exercise (30 watt) in the supine position under an intra-venous infusion of N^G-monomethyl-L-arginine (L-NMMA: 3 mg[[rad]]kg^-1 during the initial 5 min and subsequent continuous infusion of 50µg[[rad]]kg^-1[[rad]]min^-1 in saline) or vehicle (saline) at random order on separate days. The pulse wave velocity (PWV) from the femoral to posterior tibial artery was measured on both legs before and after the infusion at rest, and 2 min after the exercise. Under the control condition, the exercised leg PWV significantly decreased after the exercise (P<0.05), whereas the non-exercised leg PWV did not show a significant change throughout the experiment. Under the L-NMMA administration condition, the exercised leg PWV significantly increased by the infusion (P<0.05) but significantly decreased after the exercise (P<0.05). The non-exercised leg PWV increased with the L-NMMA administration and maintained significantly higher level over the administration compared with the baseline (before the infusion, all P<0.05). The NOS blockade x time interaction on the exercised leg PWV was not significant (P=0.706). These results suggest that the increased production of NO is not a major factor in the decrease of the regional arterial stiffness with low-intensity, short-duration aerobic exercise.