Hepatocellular carcinoma (HCC) is the most common type of liver cancer worldwide, and it is the second leading cause of cancer-related mortality. Aquaporin 9 (AQP9) is an essential aquaporin in the liver and located in the basolateral membrane of hepatocytes, but its roles on HCC has not been completely elucidated. This study investigated the regulatory functions of AQP9 in the pathogenesis of HCC. The expression levels of AQP9 were significantly down-regulated in HCC tissues and cells, which was also correlated with tumor size and number, TNM stage, five-year survival rate, lymphatic and distal metastasis within the patients. Furthermore, overexpressed AQP9 suppressed the proliferation, migration and invasion of HCC cells. The levels of PCNA, E-cad, N-cad, α-SMA, DVL2, GSK-3β, cyclinD1 and β-catenin in HCC cells were reduced by overexpressed AQP9, while cell apoptosis was remarkably enhanced. Additionally, following the treatment with Wnt/β-catenin signaling inhibitor (XAV939), the proliferative activity of HCC cells was significantly inhibited; PCNA and EMT-related markers were down-regulated; migration and invasion of cells were notably suppressed; cell apoptotic rate was decreased. Vice versa, after the cells were treated with Wnt/β-catenin inducer (SKL2001), the effects caused by overexpressed AQP9 were abrogated. In vivo studies indicated that tumor volume and weight were remarkably decreased in AQP9 overexpression group, where the levels of Wnt/β-catenin signaling- and EMT-associated molecules were also reduced. Taken together, our results suggested that overexpressed AQP9 could inhibit growth and metastasis of HCC cells via Wnt/β-catenin pathway. AQP9 may be a promising therapeutic target for the treatment of patients with HCC.