A growing number of human studies have reported the beneficial influences of acute as well as chronic exercise on cognitive functions. However, neuroimaging investigations into the neural substrates of the effects of acute exercise have yet to be performed. Using multichannel functional near-infrared spectroscopy (fNIRS), we sought cortical activation related to changes in the Stroop interference test, elicited by an acute bout of moderate exercise, in healthy volunteers (N=20). The compactness and portability of fNIRS allowed on-site cortical examination in a laboratory with a cycle ergometer, enabling strict control of the exercise intensity of each subject by assessing their peak oxygen intake (VO2peak). We defined moderate exercise intensity as 50% of a subject's peak oxygen uptake (50%VO2peak). An acute bout of moderate exercise caused significant improvement of cognitive performance reflecting Stroop interference as measured by reaction time. Consistent with previous functional neuroimaging studies, we detected brain activation due to Stroop interference (incongruent minus neutral) in the lateral prefrontal cortices in both hemispheres. This Stroop-interference-related activation was significantly enhanced in the left dorsolateral prefrontal cortex due to the acute bout of moderate exercise. The enhanced activation significantly coincided with the improved cognitive performance. This suggests that the left dorsolateral prefrontal cortex is likely the neural substrate for the improved Stroop performance elicited by an acute bout of moderate exercise. fNIRS, which allows physiological monitoring and functional neuroimaging to be combined, proved to be an effective tool for examining the cognitive effects of exercise.