Chronic stress-associated pathologies frequently associate with alterations in the structure and activity of the medial prefrontal cortex (mPFC). However, the influence of infralimbic cortex (IL) projection neurons on hypothalamic-pituitary-adrenal (HPA) axis activity is unknown, as is the involvement of these cells in chronic stress-induced endocrine alterations. In the current study, a lentiviral-packaged vector coding for a small interfering RNA (siRNA) targeting vesicular glutamate transporter (vGluT) 1 messenger RNA (mRNA) was microinjected into the IL of male rats. vGluT1 is responsible for presynaptic vesicular glutamate packaging in cortical neurons, and knockdown reduces the amount of glutamate available for synaptic release. After injection, rats were either exposed to chronic variable stress (CVS) or remained in the home cage as unstressed controls. Fifteen days after the initiation of CVS, all animals were exposed to a novel acute stressor (30-minute restraint) with blood collection for the analysis of adrenocorticotropic hormone (ACTH) and corticosterone. Additionally, brains were collected for in situ hybridization of corticotrophin-releasing hormone mRNA. In previously unstressed rats, vGluT1 siRNA significantly enhanced ACTH and corticosterone secretion. Compared with CVS animals receiving the green fluorescent protein control vector, the vGluT1 siRNA further increased basal and stress-induced corticosterone release. Further analysis revealed enhanced adrenal responsiveness in CVS rats treated with vGluT1 siRNA. Collectively, our results suggest that IL glutamate output inhibits HPA responses to acute stress and restrains corticosterone secretion during chronic stress, possibly at the level of the adrenal. Together, these findings pinpoint a neurochemical mechanism linking mPFC dysfunction with aberrant neuroendocrine responses to chronic stress.