OBJECTIVE : Obestatin regulates water metabolism by inhibiting arginine vasopressin (AVP) release and upregulated obestatin has been detected in patients with chronic heart failure (CHF). However, the significance of obestatin in CHF, particularly with regard to water retention and aquaporin 2 (AQP2) expression, remains unknown. METHODS : Using a CHF rat model, the effects of 2-week exogenous obestatin administration were evaluated. Expression of AQP2 was evaluated by immunoblotting, immunohistochemical staining, and quantitative real-time PCR (qPCR) in CHF rat model and mouse inner medullary collecting duct (mIMCD) 3 cell line. Moreover, the influence of obestatin on the genetic transcription profile in mIMCD3 cells was evaluated by microarray, and the potential regulatory mechanisms of obestatin on AQP2 were evaluated by RNA silencing of vasopressin receptor 2 (V2R), peroxisome proliferator-activated receptor gamma (PPARG), and G protein-coupled receptor 39 (GPR39). RESULTS : Obestatin increased urinary output and improved expression of CHF biomarker without significantly altering cardiac function, plasma electrolyte concentrations, or the plasma AVP concentration. AQP2 expression was significantly reduced. The results of microarray analyses and qPCR indicated that mRNA levels of Aqp2, Pparg, and V2r were significantly decreased. Inhibition of V2r and Pparg mRNA further reduced the expression of AQP2, while the inhibitory efficacy of obestatin on AQP2 was significantly offset after Gpr39 knockdown. CONCLUSIONS : Long-term treatment with obestatin improves water retention in CHF by increasing urinary output through downregulation of AQP2 expression in renal IMCD cells. These effects may be at least partially mediated by regulation of GPR39, V2R and PPARG signaling.