Abstract: There is a strict interaction between the autonomic nervous system (ANS) and pain, which might involve descending pain modulatory mechanisms. The periaqueductal grey (PAG) is involved both in descending pain modulation and ANS, but its role in mediating this relationship has not yet been explored. Here, we sought to determine brain regions mediating ANS and descending pain control associations. Thirty participants underwent conditioned pain modulation (CPM) assessments, in which they rated painful pressure stimuli applied to their thumbnail, either alone or with a painful cold contralateral stimulation. Differences in pain ratings between ‘pressure‐only’ and ‘pressure + cold’ stimuli provided a measure of descending pain control. In 18 of the 30 participants, structural scans and two functional MRI assessments, one pain‐free and one during cold‐pain were acquired. Heart rate variability (HRV) was simultaneously recorded. Normalised low‐frequency HRV (LF‐HRVnu) and the CPM score were negatively correlated; individuals with higher LF‐HRVnu during pain reported reductions in pain during CPM. PAG‐ventro‐medial prefrontal cortex (vmPFC) and PAG‐rostral ventromedial medulla (RVM) functional connectivity correlated negatively with the CPM. Importantly, PAG‐vmPFC functional connectivity mediated the strength of the LF‐HRVnu‐CPM association. CPM response magnitude was also negatively correlated with vmPFC GM volume. Our multi‐modal approach, using behavioural, physiological and MRI measures, provides important new evidence of interactions between ANS and descending pain mechanisms. ANS dysregulation and dysfunctional descending pain modulation are characteristics of chronic pain. We suggest that further investigation of body‐brain interactions in chronic pain patients may catalyse the development of new treatments. image Key points: Heart rate variability (HRV) is associated with descending pain modulation as measured by the conditioned pain modulation protocol (CPM). There is an association between CPM scores and the functional connectivity between the periaqueductal grey (PAG) and ventro‐medial prefrontal cortex (vmPFC). CPM scores are also associated with vmPFC grey matter volume. The strength of functional connectivity between the PAG and vmPFC mediates the association between HRV and CPM. Our data provide new evidence of interactions between the autonomic nervous system and descending pain mechanisms.