Non-medical prescription opioid use (NMPOU) recently increased dramatically, especially in the U.S. Although chronic opioid use is commonly accompanied by deficits in social functioning and dysregulation of the hypothalamic-pituitary adrenergic (HPA) stress axis, little is known about the impact of NMPOU on psychosocial stress responses. Therefore, we measured physiological responses of the autonomic nervous system and the HPA axis to social rejection using the Cyberball paradigm. We compared 23 individuals with NMPOU, objectively confirmed by hair and urine analyses, with 29 opioid-naive, healthy controls. As expected, heart rate variability (HRV), an index of parasympathetic activity, increased significantly during exclusion within controls, while in the NMPOU group only a trend in the same direction was found. However, increased HRV was robustly moderated by opioid craving indicating worse emotion regulation to social exclusion specifically in individuals with high opioid craving. Greater levels of the adrenocorticotropic hormone and cortisol responses to social rejection were found in the NMPOU group indicating hyperreactivity of the HPA axis to social exclusion. Self-ratings suggest that opioid users were aware of rejection, but less emotionally affected by exclusion. Furthermore, controls showed greater negative mood after the Cyberball confirming the task\'s validity. Moreover, NMPOU individuals reported a smaller social network size compared to controls. Present findings suggest that chronic NMPOU is associated with dysfunctional physiological responses to psychosocial stressors such as social rejection. In sum, NMPOU was associated with poorer regulation of the parasympathetic nervous system, especially under opioid craving highlighting its potential importance in relapse prevention.