The NADPH oxidase NOX4 has emerged as an important source of reactive oxygen species (ROS) in signal transduction, playing roles in physiological and pathological processes. NOX4 mediates Transforming Growth Factor-beta (TGF-β)-induced intracellular signals that provoke liver fibrosis and pre-clinical assays have suggested NOX4 inhibitors as useful tools to ameliorate this process. However, the potential consequences of sustained treatment of liver cells with NOX4 inhibitors are unknown yet. The aim of this work was to analyze whether NOX4 plays a role in regulating liver cell growth either under physiological conditions or during tumorigenesis. In vitro assays proved that stable knock-down of NOX4 expression in human liver tumor cells increased cell proliferation, which correlated with a higher percentage of cells in S/G2/M phases of the cell cycle, down-regulation of p21(CIP1/WAF1), increase of Cyclin D1 protein levels and nuclear localization of beta-catenin. Silencing of NOX4 in untransformed human and mouse hepatocytes also increased their in vitro proliferative capacity. In vivo analysis in mice revealed that NOX4 expression was down-regulated under physiological proliferative situations of the liver, such as regeneration after partial hepatectomy, as well as during pathological proliferative conditions, such as diethyl-nitrosamine (DEN)-induced hepatocarcinogenesis. Xenograft experiments in athymic mice indicated that NOX4 silencing conferred advantage to the human hepatocarcinoma cells, resulting in earlier onset of tumor formation and increase in tumor size. Interestingly, immunochemical analyses of NOX4 expression in human liver tumor cell lines and tissues revealed decreased NOX4 protein levels in liver tumorigenesis. Overall, results described here strongly suggest that NOX4 would play a growth inhibitory role in liver cells.