Activating transcription factor 6α (ATF6α) is a sensor of endoplasmic reticulum (ER) stress, and reportedly increases the expression of ER chaperones such as immunoglobulin heavy chain binding protein (BiP) and molecules related to the ER associated degradation of unfolded/misfolded proteins. We previously reported that BiP expression is increased in the arginine vasopressin (AVP) neurons of the supraoptic nucleus (SON) in mice subjected to dehydration. In this study, we employed ATF6α knockout (ATF6αKO) mice to clarify the role of ATF6α in the AVP neuron systems. While urine volumes were not different between ATF6αKO and wild-type (WT) mice with access to water ad libitum, they were increased in ATF6αKO mice than in WT mice under intermittent water deprivation (WD; for 48 h once a week), accompanied by less urine AVP in ATF6αKO mice compared to WT mice. BiP mRNA expression in the SON was significantly increased in WT mice but not in ATF6αKO after WD for 12 weeks. Electron microscopic analyses demonstrated that the ER lumen of AVP neurons was more dilated in ATF6αKO mice compared to WT mice after WD. We also examined the phenotypes ATF6αKO mice mated with mice possessing a mutation causing familial neurohypophysial diabetes insipidus (FNDI), which are characterized by progressive polyuria and AVP neuronal loss due to the accumulation of mutant AVP precursor in the ER. Urine volumes of FNDI/ATF6αKO mice significantly increased compared to FNDI mice after WD, accompanied by less urine AVP in FNDI/ATF6αKO mice compared to FNDI mice. The aggregate formation in the ER of AVP neurons was further hampered in FNDI/ATF6αKO mice compared to FNDI mice, and AVP neuronal loss was accelerated in the FNDI/ATF6αKO mice after WD for 12 weeks. These data suggest that ATF6α is required for the maintenance of ER function in AVP neurons under dehydration.