The brain's low resistance ensures a robust blood flow throughout systole and diastole and is susceptible to flow pulsatility. Increased cerebral pulsatility contributes to the progression of cerebrovascular disease. Although aerobic exercise affects vascular function, little is known about the effect of exercise on the cerebral pulsatility index in older adults. The aim of this study was to investigate the effect of exercise training on the post-exercise cerebral pulsatility response in older adults. Ten healthy older adults participated in a 12-week exercise training intervention. Before and after the intervention, we measured the pulsatility index of the middle cerebral artery by means of transcranial Doppler method at baseline and following a cycling exercise bout performed at an intensity corresponding to the ventilatory threshold. Before exercise training, there was no significant change in the cerebral pulsatility response to an acute bout of cycling exercise. However, after the intervention, cerebral pulsatility decreased significantly following 30 min of an acute cycling exercise (P < 0.05). This study demonstrated that cerebral pulsatility index did not change following an acute bout of cycling exercise at an intensity corresponding to ventilatory threshold, but that, after 12 weeks of exercise training, cerebral pulsatility index was reduced at 30 min after a single bout of cycling exercise. These results suggest that long-term aerobic exercise training may enhance the post-exercise reduction in pulsatility index in older adults.