Guilt, shame, and embarrassment are quintessential moral emotions with important regulatory functions for the individual and society. Moral emotions are, however, difficult to study with neuroimaging methods because their elicitation is more intricate than that of basic emotions. Here, using functional MRI (fMRI), we employed a novel social prejudice paradigm to examine specific brain regions associated with real-time moral emotion, focusing on guilt and related moral-negative emotions. The paradigm induced intense moral-negative emotion (primarily guilt) in 22 low-prejudice individuals through preprogrammed feedback indicating implicit prejudice against Black and disabled people. fMRI data indicated that this experience of moral-negative emotion was associated with increased activity in anterior paralimbic structures, including the anterior cingulate cortex (ACC) and anterior insula, in addition to areas associated with mentalizing, including the dorsomedial prefrontal cortex, posterior cingulate cortex, and precuneus. Of significance was prominent conflict-related activity in the supragenual ACC, which is consistent with theories proposing an association between acute guilt and behavioral inhibition. Finally, a significant negative association between self-reported guilt and neural activity in the pregenual ACC suggested a role of self-regulatory processes in response to moral-negative affect. These findings are consistent with the multifaceted self-regulatory functions of moral-negative emotions in social behavior.