Although numerous neuroimaging studies have examined what happens when individuals are instructed to regulate their emotions, we rarely receive such instruction in everyday life. This study sought to examine what underlies uninstructed modulation of negative affect by examining neural responses when ‘responding naturally’ to negative stimuli—and for comparison—during instructed reappraisal of negative stimuli as well. Two analyses were conducted to identify how variability in negative affect related to neural responses when responding naturally. First, in a within-participant analysis, lower levels of self-reported negative affect on a given trial were associated with recruitment of dorsolateral and dorsomedial prefrontal cortex (PFC)—brain regions also active during instructed reappraisal—whereas higher levels of negative affect were associated with recruitment of the amygdala—a region that responded more strongly overall to negative than neutral stimuli. Second, in a between-participant analysis, lower levels of average self-reported negative affect were associated with recruitment of ventromedial PFC. These results suggest that uninstructed modulation of emotion involves a combination of two types of regulatory processes, with moment-to-moment modulation depending on prefrontal regions that support reappraisal and individual differences in modulation depending on ventromedial PFC, a region involved in fear extinction.