Information on cerebral oxygenation during prolonged driving in healthy humans may help to explain the cause and development of central fatigue and its effects on cortex activities. The objective of this study is to investigate the time course of cerebral oxygenation during a prolonged driving task. Forty healthy male subjects were randomly divided into two groups: task group (Task) and control group (CNL). All subjects were required to rest well prior to the experiment. For the task group, subjects were required to perform the simulated driving task for 3 h. Cerebral oxygenation signal was monitored from the left frontal lobe using near infrared spectroscopy throughout the entire experiment. Significant increases in the concentrations of HbO(2) (DeltaCHbO(2)) and HbT (DeltaCHbT) were recorded at the start of driving task compared with the resting value (p < 0.01). The cerebral oxygen saturation in the Task group was found to be significantly lower following three hours of driving compared with that in the CNL (F = 16.95, p < 0.001). In addition, a significant difference in selective reaction time was observed between the Task group and CNL during the post-task period (p = 0.023). The results demonstrated that the cerebral oxygenation is closely related to the mental stress. The decrease in the cerebral oxygen saturation may indicate reduced cerebral oxygen delivery, and this may be an important factor affecting central fatigue development during prolonged driving.