Emotional neuroscience maps neurocircuits associated with the processing of affective stimuli. To assess gender differences in brain activation elicited by affective stimuli, we used pictures from the International Affective Picture System in a functional magnetic resonance imaging (fMRI) study. Ten male and ten female age-matched healthy volunteers were included and viewed affectively negative versus positive pictures, which were presented in an event related design. There was a significant interaction between valence of emotional stimuli and gender in the sublenticular extended amygdala (SLEA) and the rostral anterior cingulate. fMRI activation in these regions was stronger for negative compared to positive cues in women. In men fMRI activation was independent of stimulus valence. These results suggest to take gender differences into account when emotional paradigms are tested in functional brain imaging.