© 2016 Society of Biological Psychiatry. Background Anxiety is the most common comorbid psychiatric concern in individuals diagnosed with autism spectrum disorder (ASD) and can cause significant functional impairment. Fear conditioning tasks offer a useful neurodevelopmental model for anxiety, yet there are no published neuroimaging studies of fear conditioning using ASD samples. Methods Twenty adults diagnosed with ASD and 19 healthy adult control subjects completed a standard fear conditioning and extinction paradigm while undergoing functional magnetic resonance imaging scanning. A burst of air on the base of the neck was the unconditioned stimulus. Participants returned 1 day later for scanning during an extinction recall phase. A priori regions of interest were analyzed with a familywise error correction rate < 0.05. Results All regions of interest demonstrated significantly greater response to threat than safe conditions during initial fear acquisition. Compared with age-matched control subjects, the ASD group showed a significantly decreased differential response to threat versus safe cues in right amygdala during the initial fear acquisition phase and decreased response in left amygdala during the first run of extinction recall on the second day of scanning. Conclusions Symptoms of severe anxiety in ASD may arise from atypical neural mechanisms especially related to the differentiation of threat versus safe cues. An inability to effectively identify safety contexts may underlie chronically increased levels of anxiety in many individuals diagnosed with ASD.