Chronic stress and chronic pain exacerbate one another and worsen outcomes in clinical populations. The anatomical locations where neurophysiological changes underlying chronic stress and pain comorbidity could occur are poorly explored. In this study, we implemented a mouse model of chronic unpredictable stress (CUS) to test the effects of established stress on reflexive and nonreflexive pain behaviors and the ability to recover from painful neuropathy and post-operational injury. We further examined the effects of stress on neuronal structure and function in a subregion of the medial prefrontal cortex, the prelimbic cortex (PL), an area implicated in both stress and pain. CUS induced thermal hypersensitivity, mechanical allodynia, and reduced pain tolerance in male, but not in female, mice. Stressed male mice also showed persistent hypersensitivity and anxiety-like behavior compared to controls following chemotherapy and paw incision injuries. cFos expression in PL following an acute noxious stimulus was reduced in CUS mice indicating reduced prefrontal activity. However, PL layer V neurons that project to the ventrolateral periaqueductal gray (vlPAG) did not show changes in density of dendritic spines in distal branches of the apical dendrite, nor did they show changes in intrinsic membrane excitability following CUS. In contrast, CUS did produce increased spontaneous inhibitory drive onto PL-vlPAG neurons, altering the excitatory to inhibitory ratio. Our results suggest that stress and pain work in conjunction to promote persistent hypersensitivity and negative affective behaviors, and provide evidence that stress increases inhibitory synaptic transmission onto mPFC-vlPAG descending projection neurons. Perspective: Chronic unpredictable stress produced hypersensitivity and worsened outcomes after a painful injury in male mice. The prelimbic cortex is identified as an important region where chronic stress may modulate pain. We demonstrate a clinically relevant model that can be used to investigate neural correlates underlying stress and pain interactions.