Corticotropin-releasing factor (CRF) isthekeyregulatorofthehypothalamic-pituitary-adrenalaxis.CRF neurons cannot be distinguished morphologically from other neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) without immunostaining. Thus, we generated a knock-in mouse that expresses modified yellow fluorescent protein (Venus) in CRF neurons (CRF-Venus), andyet its expression is drivenbytheCRFpromoterandresponds tochangesin the interior milieu. In CRF-Venus, Venus-expressing neurons were distributed in brain regions harboring CRF neurons, including thePVH.Themajority of Venus-expressing neurons overlapped with CRF-expressing neurons in the PVH, but many neurons expressed only Venus or CRF in a physiological glucocorticoid condition. After glucocorticoid deprivation, however, Venus expression intensified, and most Venus neurons coexpressed CRF. Conversely, Venus expression was suppressed by excess glucocorticoids. Expression of copeptin, a peptide encoded within the vasopressin gene, was induced in PVH-Venus neurons by glucocorticoid deprivation and suppressed by glucocorticoid administration. Thus, Venus neurons recapitulated glucocorticoid-dependent vasopressin expression in PVH-CRF neurons. Noradrenaline increased the frequency of glutamate-dependent excitatory postsynaptic currents recorded from Venusexpressing neurons in the voltage clamp mode. In addition, the CRF-iCre knock-in mouse was crossed with a CAG-CAT-EGFP reporter mouse to yield the TgCAG-CAT-EGFP/wt;CRFiCre/wt(EGFP/CRF-iCre) mouse, inwhichenhancedgreenfluorescentprotein(EGFP)isdrivenbytheCAGpromoter.EGFPwasexpressed more constitutively in the PVH of EGFP/CRF-iCre mice. Thus, CRF-Venus may have an advantage for monitoring dynamic changes in CRF neurons and CRF networks in different glucocorticoid states.