Increasing evidence shows that the generation and regulation of affective responses is associated with activity of large brain networks that also include phylogenetically older regions in the brainstem. Mesencephalic regions not only control autonomic responses but also participate in the modulation of autonomic, emotional, and motivational responses. The specific contribution of the midbrain to emotion regulation in humans remains elusive. Neuroimaging studies grounding on appraisal models of emotion emphasize a major role of prefrontal cortex in modulating emotion-related cortical and subcortical regions but usually neglect the contribution of the midbrain and other brainstem regions. Here, the role of mesolimbic and mesocortical networks in core affect generation and regulation was explored during emotion regulation guided by real-time fMRI feedback of the anterior insula activity. The fMRI and functional connectivity analysis revealed that the upper midbrain significantly contributes to emotion regulation in humans. Moreover, differential functional interactions between the dopaminergic mesocorticolimbic system and frontoparietal networks mediate up and down emotion regulatory processes. Finally, these findings further indicate the potential of real-time fMRI feedback approach in guiding core affect regulation.