BACKGROUND: Chronic alcohol consumption in alcohol use disorder (AUD) is associated with autonomic nervous system dysregulation, increasing cardiovascular risk, and high alcohol cravings. Heart rate variability (HRV), a marker of autonomic nervous system responsiveness to stressors, may mediate alcohol's impact on the cardiovascular system. While pregnenolone (PREG) has been shown to normalize heart rate and blood pressure in individuals with AUD, its effects on sympathetic and parasympathetic components of HRV and related alcohol craving are not known. METHODS: Fifty-five treatment-seeking individuals with AUD were randomized to placebo (n = 21) or daily pregnenolone at 300 mg (n = 18) or 500 mg (n = 16), in a double-blind, 8-week pilot clinical trial. In week 2, participants underwent three randomized, counterbalanced 5-minute personalized guided imagery provocations (stress, alcohol, and neutral/relaxing cues) on separate days. HRV indices were assessed during each session and analyzed using linear mixed-effects models (LMEs), including association between HRV indices and anxiety and alcohol craving. RESULTS: A medication group × condition interaction was found for parasympathetic, high-frequency (HF) (p = 0.028) and sympathetic/parasympathetic, low-frequency/high-frequency (LF/HF) ratio (p = 0.017) indices of HRV. Placebo had higher HF during alcohol cue (p = 0.011), while 500 mg PREG demonstrated lower HF in response to stress (p = 0.050) and alcohol cues (p = 0.047). Placebo showed lower LF/HF ratio during stress (p = 0.006) and alcohol cue (p = 0.001), while the PREG groups showed no changes. Overall, the LF/HF response to alcohol cue was significantly lower in placebo compared to the 300 mg PREG (p = 0.012) and 500 mg PREG (p = 0.037) groups. Lastly, HF was found to predict alcohol craving regardless of PREG doses. CONCLUSIONS: We found a normalization of autonomic response in PREG groups. These findings suggest that PREG holds therapeutic potential for enhancing autonomic function in AUD.