Presentation of negative pictures was used as emotional stress to assess gender differences in prefrontal area activation in a functional near-infrared spectroscopy (NIRS) study. Compared with neutral condition, the response of oxy-HB for men yielded no significant difference during stress period, but the response induced by stress pictures for women showed significant enhancement. It was indicated that it is crucial to take gender difference into account when negative stimuli are used in functional brain imaging.