Glucocorticoid (GC) production is physiologically regulated through a negative feedback loop mediated by the GC, which appear disrupted in several pathological conditions. The inability to perform negative feedback of the hypothalamus-pituitary-adrenal (HPA) axis in several diseases is associated with an overproduction of reactive oxygen species (ROS); however, nothing is known about the effects of ROS on the functionality of the HPA axis during homeostasis. This study analyzed the putative impact of antioxidants on the HPA axis activity and GC-mediated negative feedback upon the HPA cascade. Male Wistar rats were orally treated with N-acetylcysteine (NAC) or vitamin E for 18 consecutive days. NAC-treated rats were then subjected to a daily treatment with dexamethasone, which covered the last 5 days of the antioxidant therapy. We found that NAC and vitamin E induced an increase in plasma corticosterone levels. NAC intensified MC2R and StAR expressions in the adrenal and reduced GR and MR expressions in the pituitary. NAC also prevented the dexamethasone-induced reduction in plasma corticosterone levels. Furthermore, NAC decreased HO-1 and Nrf2 expression in the pituitary. These findings show that antioxidants induce hyperactivity of the HPA axis via upregulation of MC2R expression in the adrenal and downregulation of GR and MR in the pituitary.