Needle procedures are common throughout childhood and often elicit distress in children and parents. Heart rate variability (HRV), as an index of emotion regulation, can inform both self-regulatory and co-regulatory processes. Mindfulness may serve to regulate distress; however, no research has studied mindfulness or parent and child regulatory responding concurrently during venipuncture. Stemming from a randomized controlled trial investigating a mindfulness intervention, this study sought to describe regulatory responding (via HRV) throughout pediatric venipuncture and the role of cognitive-affective factors (mindfulness, parent anxiety, catastrophizing) in 61 parent-child dyads (7-12 years). We examined (1) patterns of parent and child HRV throughout venipuncture and whether a brief, randomly assigned audio-guided mindfulness versus control exercise affected this pattern and (2) the extent to which changes in parent and child HRV were synchronized throughout venipuncture, and whether parent catastrophizing and anxiety moderated this association. HRV differed as a function of procedural phase. Practicing the mindfulness versus control exercise did not consistently affect HRV in dyads. Positive synchrony was observed during the end of the intervention in dyads with high parental catastrophizing. Otherwise, a pattern of nonsynchrony emerged. Results provide foundational knowledge regarding children's internal (self) and external (parent) regulation mechanisms. RCT registration: NCT03941717.