Endoplasmic reticulum (ER) stress has been implicated in some types of glomerular and tubular disorders. The objectives of this study were to elucidate the role of ER stress in lithium-induced nephrogenic diabetes insipidus (NDI) and to investigate whether attenuation of ER stress by 4-phenybutyric acid (4-PBA) improves urinary concentrating defect in a lithium-treated rats. Wistar rats have received lithium (40 mmol/kg food), 4-PBA (320 mg/kg BW by gavage every day), or no treatment (control) for two weeks and some of them were dehydrated for 24 hours. Lithium treatment resulted in increased urine output and decreased urinary osmolality, which was significantly improved by 4-PBA. 4-PBA also prevented reduced protein expression of AQP2, pS256-AQP2, and pS261-AQP2 in inner medullar of kidneys from lithium-treated rats after 24 hour dehydration. Lithium treatment resulted in increased expression of ER stress markers in inner medulla, which was associated with dilated cisternae and expansion of ER in the inner medullary collecting duct (IMCD) principal cells. Confocal immunofluorescence studies showed colocalization of a molecular chaperone BiP with AQP2 in principal cells. Immunohistochemistry demonstrated increased intracellular expression of BiP and decreased AQP2 expression in IMCD principal cells of kidneys from lithium-treated rats. 4-PBA attenuated expression of ER stress markers and recovered ER morphology. In IMCD suspensions isolated from lithium-treated rats, 4-PBA incubation was associated with increased AQP2 expression and ameliorated ER stress. In conclusion, in experimental lithium-induced NDI, 4-PBA improved urinary concentrating defect and increased AQP2 expression, likely via attenuating ER stress in inner medullary collecting duct principal cells.