Rationale <p>Negative affect plays a prominent role in the maintenance of alcohol use disorder (AUD) and has been identified as a risk factor for relapse to alcohol. To date, however, treatment options that target negative affective states and consecutive relapse risk in AUD are insufficient. Oxytocin (OXY) might be a promising approach for addressing negative affective states and resulting motivation to use alcohol.</p> Objectives <p>We aimed to investigate the acute effects of 24 I.U. OXY, administered intranasally, compared to matched placebo (PLC) on central processing of negative emotional stimuli in the amygdala in individuals with AUD.</p> Methods <p>We conducted a randomized double-blind placebo-controlled crossover study in <i>N</i> = 24 individuals with AUD. Amygdala response to emotional stimuli served as primary outcome and was assessed using a validated functional magnetic resonance imaging emotion-processing task. Alcohol craving served as secondary outcome.</p> Results <p>OXY versus PLC attenuated right amygdala reactivity to fearful and angry emotional face stimuli during the fMRI task (<i>t</i>(33) = 3.32, <i>p</i>_FWE=0.035), while no effect of OXY on amygdala activation was observed during the presentation of geometric figures. In addition, right amygdala reactivity to fearful and angry emotional face stimuli was positively associated with alcohol craving (<i>r</i> =.332, Bias corrected and accelerated 95% confidence interval [95% BCa CI]=-0.044 to 0.624, <i>p</i> =.042).</p> Conclusions <p>OXY’s effects on the neurocircuitry underlying negative affect and craving in AUD support its potential for dampening alcohol craving induced by negative affective states and implicate OXY as a potential future treatment option for AUD.</p> Clinicaltrials registry <p>DRKS00026218.</p>