To monitor the cerebral cortical activation in healthy subjects and stroke patients during walking under virtual reality (VR) environment in real-time using functional near-infrared spectroscopy (fNIRS), and to preliminarily explore the changes in brain activity of stroke patients under VR gait rehabilitation training. Methods: A total of 17 stroke patients and 17 healthy volunteers were recruited and assigned to the stroke group and the control group, respectively. The Berg Balance Scale (BBS) was used to assess the balance function of the subjects, and the Timed Up and Go Test (TUGT) was used to evaluate their walking function. The fNIRS was employed to collect data on the concentration of oxyhemoglobin (HbO) in the brain regions of the two groups of subjects at rest, during stepping in a general environment, and during stepping in a VR environment. The correlation between the significantly activated channels in the brain regions of the two groups and the scores of BBS and TUGT was analyzed. Results: (1) At rest, compared with the control group, the stroke group had a lower HbO concentration in the left sensorimotor cortex (P< 0.05) and a higher HbO concentration in the right sensorimotor cortex (P<0.05). (2) During the general stepping task, the activation of the right motor cortex decreased in stroke patients (P<0.05), while the activation of the left sensory cortex increased (P<0.05). In the VR stepping task, the activation of the left prefrontal cortex increased in stroke patients (P<0.05). (3) Spearman correlation analysis of the channels with significant differences in brain region activation between the two groups and the total scores of Berg and TUGT scores showed that in the VR stepping task, the activation of the prefrontal cortex (ch9) was negatively correlated with the total scores of Berg (r=－0.521, P=0.003), and positively correlated with the TUGT scores (r=0.434, P=0.017). Conclusion: The mechanism by which VR affects the walking function of stroke patients may be related to changes in the activation of the prefrontal cortex.