Despite mounting reports about the negative effects of chronic occupational stress on cognitive and emotional functions, the underlying mechanisms are unknown. Recent findings from structural MRI raise the question whether this condition could be associated with a functional uncoupling of the limbic networks and an impaired modulation of emotional stress. To address this, 40 subjects suffering from burnout symptoms attributed to chronic occupational stress and 70 controls were investigated using resting state functional MRI. The participants' ability to up- regulate, down-regulate, and maintain emotion was evaluated by recording their acoustic startle response while viewing neutral and negatively loaded images. Functional connectivity was calculated from amygdala seed regions, using explorative linear correlation analysis. Stressed subjects were less capable of down-regulating negative emotion, but had normal acoustic startle responses when asked to up-regulate or maintain emotion and when no regulation was required. The functional connectivity between the amygdala and the anterior cingulate cortex correlated with the ability to down-regulate negative emotion. This connectivity was significantly weaker in the burnout group, as was the amygdala connectivity with the dorsolateral prefrontal cortex and the motor cortex, whereas connectivity from the amygdala to the cerebellum and the insular cortex were stronger. In subjects suffering from chronic occupational stress, the functional couplings within the emotion- and stress-processing limbic networks seem to be altered, and associated with a reduced ability to down-regulate the response to emotional stress, providing a biological substrate for a further facilitation of the stress condition.