Aquaporins (AQPs) are a family of proteinaceous water channels involved in bile production and apoptosis. Aquaporins 8, 9 and 0 are expressed in hepatocytes and cAMP dependent signalling regulates AQP8 expression and localization in normal hepatocytes. Also, AQP9 has promoter binding sites for AP-1, a downstream transcription factor in IL-6 signaling. The role of AQP8 and 9 in hepatocellular carcinoma (HCC) initiation and progression has not been determined. The role of AQP8/9 in HCC initiation and progression was examined in rodent models of HCC as well as human HCC. Further, the role of cAMP and IL6 in regulating AQP8/9 expression and/or localization was examined. To examine the role of AQP8/9 in HCC initiation and progression both a rat, hepatoma cell inoculation model and a diethylnitrosamine (DEN) initiated, hepatocarcinogenic, progressive mouse model, were used. A rat hepatoma cell line was used to examine the role of cAMP and IL6 on AQP8/9 expression and localization. While IL6 significantly affected AQP8 membrane localization, cAMP did not. Further, there was no significant affect on AQP8/9 expression from cAMP pathway modulation or IL6 exposure. However, cAMP pathway modulation did significantly affect cell responsiveness to osmotic challenge. In a mouse model of HCC, AQP8 expression in plasma membrane was significantly higher in mice euthanized 24 weeks after DEN injection compared to control mice. However, AQP8 and 9 membrane localization was significantly lower in mice euthanized 48 weeks after DEN injection compared to control mice. In human HCC, AQP9 membrane localization is significantly decreased in tumor tissue compared to non-tumor tissue. Also, AQP8/9 expression did not correlate with known risk factors for HCC. Lastly, doxycycline controlled expression of AQP8, in a rat model of HCC, significantly inhibited tumor progression in vivo. In summary, AQP8 membrane localization is affected by IL6 exposure but cAMP significantly affects cell responsiveness to osmotic stress. Also, AQP9 membrane expression is reduced in rodent models of HCC as well as human HCC. Finally, expression of AQP8 in a rat model HCC, in vivo, reduced tumor proliferation.