Whether glucocorticoids mediate medial prefrontal cortex (mPFC) regulation of the amygdala in humans remains unclear. In the current study we investigated whether cortisol levels under relatively stress-free circumstances are related to amygdala resting-state functional connectivity with the mPFC. Resting-state fMRI data were acquired from 20 healthy male participants. Salivary cortisol was sampled at multiple times throughout the experiment. The cortisol area under the curve increase (AUCi) was calculated as a measure of cortisol dynamics. Next, seed based correlations were employed on the resting-state fMRI data to reveal regions of amygdala functional connectivity related to variations in cortisol AUCi. The resulting statistical maps were corrected for multiple comparisons using cluster based thresholding (Z > 2.3, p < .05). Two regions in the mPFC showed decreasing negative functional connectivity with the amygdala when a lesser decrease in cortisol AUCi was observed: the perigenual anterior cingulate cortex and medial frontal pole (BA10). Although we initially showed a relation with cortisol AUCi, it seemed that the baseline cortisol levels were actually driving this effect: higher baseline cortisol levels related to stronger negative functional connectivity with the mPFC. Endogenous cortisol levels may modulate amygdala functional connectivity with specific regions in the mPFC, even under relatively stress-free circumstances. Our results corroborate previous findings from both animal and human studies, suggesting cortisol-mediated regulation of the amygdala by the mPFC. We propose that through this feedback mechanism the stress response might be adjusted, pointing to the putative role of cortisol in modulating stress- and, more generally, emotional responses.