Abstract: Aquaporin 9 (AQP9) is a water channel membrane protein also permeable to small solutes such as urea, glycerol and 5-fluorouracil, a chemotherapeutic agent. The objective of this study were to determine the cellular and subcellular localization of AQP9 in different organs of wild and AQP9 knockout mice (males and females) by immunohistochemistry ( IHC), immunofluorescence (IF) and RT-PCR. Twenty wild (normal control) and homozygous AQP9 KO-mice (C57 BL /6J) of both sexes were used in this study. Immunocytochemical analyses using rabbit- affinity-purified anti-rat AQP9 and Rabbit EnVision as primar and secondary antibodies respectively, revealed AQP9 specific labeling in the hepatocytes, kidney, spleen and epididymis of wild type mice (AQP9⁺/⁺), but a complete absence of labeling in AQP9 ^�/� mice. In liver, the labeling was strongest at the sinusoidal surface and there was little intracellular labeling. AQP9 expression was found to be sex-linked. In female�s liver the expression of AQP9 was mostly confined to perivascular hepatocytes, whereas males showed a more homogeneous hepatocyte staining. Confocal immunofluorescence confirmed the localization of AQP9 immunostaining on the sinusoidal surface or basolateral plasma membrane of hepatocytes. In the absence of physiological stress, knockout mice did not display any visible behavioral or severe physical abnormalities. Compared with control mice, serum levels of glycerol and triglycerides were significantly increased, in association with hypoglycemia in the AQP9^�/� mice, whereas total cholesterol, urea, alanine aminotransferase and alkaline phosphatase were not statistically different. These findings showed that AQP9 is a major water channel protein that is expressed throughout different organs, with high expression in the liver of male mice. Moreover, AQP9 is important for hepatic glycerol metabolism.