One foundational distinction in affective science is between emotion reactivity and regulation. This conceptual distinction has long been assumed to be instantiated in spatially separable brain systems (a typical example: amygdala/insula for reactivity and frontoparietal areas for regulation). In this research, we begin by reviewing previous findings that support and contradict the neural separability hypothesis concerning emotional reactivity and regulation. Further, we conduct a direct test of this hypothesis with empirical data. In five studies involving healthy and clinical samples (total n = 336), we assessed neural responses using fMRI while participants were asked to either react naturally or regulate their emotions (using reappraisal) while viewing emotionally evocative stimuli. Across five studies, we failed to find support for the neural separability hypothesis. In univariate analyses, both presumptive “reactivity” and “regulation” brain regions demonstrated equal or greater activation for the reactivity contrast than for the regulation contrast. In multivariate pattern analyses (MVPA), classifiers decoded reactivity (vs. neutral) trials more accurately than regulation (vs. reactivity) trials using multivoxel data in both presumptive “reactivity” and “regulation” regions. These findings suggest that emotion reactivity and regulation—as measured via fMRI—may not be as spatially separable in the brain as previously assumed. Our secondary whole-brain analyses revealed largely consistent results. We discuss the two theoretical possibilities regarding the neural separability hypothesis and offer thoughts for future research.