The neural bases of the association between negative affectivity and self-focus remain unknown in healthy subjects. Building on the role of the cortical midline structures (CMS) in self-referential processing, we hypothesized that negative affectivity in healthy subjects would be associated with an increased activation of the CMS during self-referential processing. We presented positive and negative pictures to 45 healthy subjects during fMRI and asked them to judge whether the pictures were related to themselves or not (self condition), or whether the pictures were positive or negative (general condition). Negative affectivity was measured by the level of harm avoidance (HA) with the Temperament and Character Inventory. Self-referential processing activated the CMS, including the dorsal and ventral medial prefrontal cortex (MPFC) and the posterior cingulate cortex (PCC). A higher HA score was associated with a greater activation of the dorsal MPFC and PCC during self-referential processing, this greater activation being more pronounced for negative pictures in the dorsal MPFC. This increased activation of the CMS may embody the association between negative affectivity and self-focus in healthy subjects, as previously observed in major depression. Within the CMS, the dorsal MPFC may play a key role in negative affectivity, integrating an increased attention to negative stimuli with an increased attention to the self.