Heart rate variability (HRV) has been separately shown to be associated with ASD symptomatology, psychological wellbeing and emotion regulation (ER) in specific samples consisting of either individuals with ASD, those without ASD, or combined. However, no study has examined these constructs together or incorporated habitual ER strategy use. Hence, the aim of this study was to examine the relationships between resting HRV, ASD symptomatology, ER strategy use (reappraisal and suppression), and psychological wellbeing (anxiety, depression and positive wellbeing) in a combined sample of adults with and without ASD. Twenty-four adults with ASD (Mage = 31.36; SDage = 14.84) and twenty without ASD (Mage = 35.45; SDage = 12.19) completed the ER Questionnaire (ERQ), Diagnostic and Statistical Manual of Mental Disorders-5 Cross-cutting Dimensional Scale, Patient Health Questionnaire-9, Warwick-Edinburgh Mental Well-being Scale, and Autism-Spectrum Quotient-Short. Participants' resting HRV data were also collected via short-term electrocardiogram. Self-reported use of reappraisal was associated with higher resting HRV. Additionally, reappraisal predicted variance in all three HRV indices above and beyond ASD symptomatology and medication use. These preliminary findings can inform the design of future studies to determine the extent to which reappraisal impacts autonomic flexibility.