Excessive stress exposure often leads to emotional dysfunction, characterized by disruptions in healthy emotional learning, expression, and regulation processes. A prefrontal cortex (PFC)-amygdala circuit appears to underlie these important emotional processes. However, limited human neuroimaging research has investigated whether these brain regions underlie the altered emotional function that develops with stress. Therefore, the present study used functional magnetic resonance imaging (fMRI) to investigate stress-induced changes in PFC-amygdala function during Pavlovian fear conditioning. Participants completed a variant of the Montreal Imaging Stress Task (MIST) followed (25 min later) by a Pavlovian fear conditioning task during fMRI. Self-reported stress to the MIST was used to identify three stress-reactivity groups (Low, Medium, and High). Psychophysiological, behavioral, and fMRI signal responses were compared between the three stress-reactivity groups during fear conditioning. Fear learning, indexed via participant expectation of the unconditioned stimulus during conditioning, increased with stress reactivity. Further, the High stress-reactivity group demonstrated greater autonomic arousal (i.e., skin conductance response, SCR) to both conditioned and unconditioned stimuli compared to the Low and Medium stress-reactivity groups. Finally, the High stress group did not regulate the emotional response to threat. More specifically, the High stress-reactivity group did not show a negative relationship between conditioned and unconditioned SCRs. Stress-induced changes in these emotional processes paralleled changes in dorsolateral, dorsomedial, and ventromedial PFC function. These findings demonstrate that acute stress facilitates fear learning, enhances autonomic arousal, and impairs emotion regulation, and suggests these stress-induced changes in emotional function are mediated by the PFC.