The median eminence (ME) anatomically consists of external (eME) and internal (iME) layers. The hypothalamic neurosecretory cells terminate their axons in the eME and secrete their neurohormones regulating anterior pituitary hormone secretion involved in stress responses into the portal vein located in the eME. Magnocellular neurosecretory cells (MNCs) which produce arginine vasopressin (AVP) and oxytocin in the paraventricular (PVN) and supraoptic nuclei (SON) terminate their axons in the posterior pituitary gland (PP) through the iME. Here, we provide the first evidence that oestrogen modulates the dynamic changes in AVP levels in the eME axon terminals in female rats, using AVP-eGFP and AVP-DREADDs transgenic rats. Strong AVP-eGFP fluorescence in the eME was observed at all oestrus cycle stages in adult female rats but not in male transgenic rats. AVP-eGFP fluorescence in the eME was depleted after bilateral ovariectomy but re-appeared with high-dose 17β-oestradiol. AVP-eGFP fluorescence in the MNCs and PP did not change significantly in most treatments. Peripheral clozapine-N-oxide administration induced AVP-DREADDs neurone activation, causing a significant increase in plasma corticosterone levels in the transgenic rats. These results suggest that stress-induced activation of the hypothalamic-pituitary-adrenal axis may be caused by oestrogen-dependent upregulation of AVP in the eME of female rats.