Mindfulness training ameliorates clinical and self-report measures of depression and chronic pain, but its use as an emotion regulation strategy – in individuals who do not meditate – remains understudied. As such, whether it (a) down-regulates early affective brain processes and (b) depends on cognitive control systems remains unclear. We exposed meditation-naïve participants to two kinds of stimuli: negative vs. neutral images and painful vs. warm temperatures. On alternating blocks, we asked participants to either react naturally or exercise mindful-acceptance. Emotion regulation using mindful-acceptance was associated with reductions in reported pain and negative affect, reduced amygdala responses to negative images, and reduced heat-evoked responses in medial and lateral pain systems. Critically, mindful-acceptance significantly reduced activity in a distributed, a-priori neurologic signature that is sensitive and specific to experimentally-induced pain. In addition, these changes occurred in the absence of detectable increases in prefrontal control systems. The findings support the idea that momentary mindful-acceptance regulates emotional intensity by changing initial appraisals of the affective significance of stimuli, which has consequences for clinical treatment of pain and emotion.