There is increasing clinical evidence that physical activity and exercise can reduce and prevent the incidence of stress-related psychiatric disorders, especially depression and anxiety. Several studies have suggested that neuronal activation of the DRN, which is the major source of serotonergic neurons in the central nervous system, is implicated in anti-depressant/anxiolytic properties. Furthermore, the incidence and symptom of depression and anxiety can be involved in the dysregulation of the hypothalamic-pituitary-adrenal axis that is initiated by the activation of corticotropin-releasing factor neurons in the PVN. It is, thus, possible that physical exercise produces its anti-depressant/anxiolytic properties by affecting these neuronal activities. However, few general concepts for the optimal regimens of physical exercise for those neuronal activities involved in anti-depressant/ anxiolytic properties have been developed. In the present study, we examined how 4-week treadmill running training with different intensities (0, 15, 25m/min; 60min/day; 5 times/week) would alter the neuronal activities in the DRN and PVN during 30-min acute treadmill running in male Wistar rats, using c-Fos immunohistochemistry. Prior to the training, we measured the neuronal activities in the DRN and PVN during 30-min acute treadmill running with each the intensity (i.e., 0, 15, 25m/min) as the baseline, and compared the neuronal activity after the training with the baseline. During acute running without the training, c-Fos expression in DRN neurons was enhanced in low speed running, and the number of c-Fos positive nuclei in the PVN was increased speed-dependently. Acute running with 4-week training increased c-Fos expression in DRN neurons, regardless of the intensity, and those responses seemed to be enhanced compared to the baseline. The PVN neurons activated speed-dependently during acute running with the training, though the activity in high speed running tended to slightly decrease. These findings suggest that 4-week treadmill running can alter the neuronal activities in the DRN and PVN during acute running according to the intensity.