BACKGROUND: Many studies employed cue exposure paradigms to investigate the neural processes underlying cue-elicited alcohol craving. Cue exposure elicits robust autonomic reactivity. However, whether or how cue-elicited autonomic response relates to the severity of alcohol misuse and the neural bases underlying the potential relationship remain unclear. METHODS: We examined cue-related brain activations in association with heart rate variability, as indexed by the root mean square of the successive differences (RMSSD), during alcohol versus neutral cue blocks in 50 adult alcohol drinkers (24 men). Imaging and heart rate variability data were collected and processed with published routines. Mediation analyses were conducted to examine the interrelationship between regional activities, cue-elicited changes in RMSSD, and the severity of problem alcohol use, as assessed with the Alcohol Use Disorders Identification Test (AUDIT). RESULTS: The results showed higher RMSSD during alcohol than during neutral cue exposures, with alcohol (vs. neutral) cue-evoked RMSSD positively correlated with AUDIT score. Further, alcohol (vs. neutral) cue-elicited activity in the ventromedial prefrontal cortex was negatively correlated both with increases in RMSSD and with the AUDIT score. Mediation analyses suggested that the RMSSD mediated the relationship between ventromedial prefrontal cortex cue activity and the AUDIT score. CONCLUSIONS: These findings substantiate the neural correlates of the presumably parasympathetic response during alcohol cue exposure and the interrelationship among ventromedial prefrontal cortex activity, autonomic response, and problem alcohol use.