Enduring sleep loss is a risk factor for a variety of both somatic and mental health issues. When subjected to sleep loss, the brain becomes vulnerable to critical alterations in cognitive and emotional processing. In our study, we examined the effect of psychosocial stress on amygdala resting-state functional connectivity in participants with cumulative sleep loss calculated across the seven days preceding scanning. For this purpose, forty-five healthy male participants completed a one-week sleep diary and underwent resting-state scans before and after taking part in the ScanSTRESS paradigm, which allows social stress induction during functional magnetic resonance imaging. Sleep loss was negatively associated with seed-based functional connectivity of the left amygdala with the medial prefrontal cortex, dorsal anterior cingulate cortex, anterior insula, posterior cingulate cortex, and dorsolateral prefrontal cortex. That is, participants with higher amounts of sleep loss showed reduced left amygdala connectivity after social stress induction to cortical regions encompassing main nodes of the brain’s default mode network and salience network. Our results shed more light on how brain functional connectivity may shape the brain’s stress response in the context of naturally occurring sleep loss, revealing a potential neural mechanism for increased vulnerability to stress-related psychopathology.