It is now well-established that stress elicits brain- and body-wide changes in physiology and has significant impacts on many aspects of health. The hypothalamic-pituitary-adrenal (HPA) axis is the major neuroendocrine system mediating the integrated response to stress. Appropriate engagement and termination of HPA activity enhances survival and optimizes physiological and behavioral responses to stress, while dysfunction of this system is linked to negative health outcomes such as depression, anxiety, and post-traumatic stress disorder. Glutamate signaling plays a large role in the transmission of stress-related information throughout the brain. Furthermore, aberrant glutamate signaling has negative consequences for neural plasticity and synaptic function and is linked to stress-related pathology. However, the connection between HPA dysfunction and glutamate signaling is not fully understood. We tested how HPA axis dysfunction (using low dose chronic corticosterone in the drinking water) affects glutamate homeostasis and neural responses under baseline and acute stress in male C57BL/6N mice. Using laser microdissection and transcriptomic analyses, we show that chronic disruption of the HPA axis alters the expression of genes related to glutamate signaling in the medial prefrontal cortex (mPFC), hippocampus, and amygdala. While neural responses to stress (as measured by FOS) in the hippocampus and amygdala were not affected in our model of HPA dysfunction, we observed an exaggerated response to stress in the mPFC. To further probe this we undertook in vivo biosensor measurements of the dynamics of extracellular glutamate responses to stress in the mPFC in real-time, and found glutamate dynamics in the mPFC were significantly altered by chronic HPA dysfunction. Together, these findings support the hypothesis that chronic HPA axis dysfunction alters glutamatergic signaling in regions known to regulate emotional behavior, providing more evidence linking HPA dysfunction and stress vulnerability.