Reactive oxygen species (ROS) play a key role in chronic liver injury and fibrosis. Homologues of NADPH oxidases (NOXs) are major sources of ROS, but the exact role of the individual homologues in liver disease is unknown. Our goal was to determine the role of NOX4 in liver fibrosis induced by bile duct ligation (BDL) with the aid of the pharmacological inhibitor GKT137831, and genetic deletion of NOX4 in mice. GKT136731 was either applied for the full term of BDL (preventive arm), or starting at 10 days post-operatively (therapeutic arm). Primary hepatic stellate cells (HSC) from control mice with and without BDL were analyzed and the effect of NOX4 inhibition on HSC activation was also studied. FasL or TNFα/actinomycin D induced apoptosis was studied in wild type and NOX4−/− hepatocytes. Results: NOX4 was upregulated by a TGF-β/Smad3-dependent mechanism in HSC. Downregulation of NOX4 decreased ROS production and the activation of NOX4−/− HSC was attenuated. NOX4−/− hepatocytes were more resistant to FasL or TNFα/actinomycin D induced apoptosis. Similarly, after pharmacological NOX4 inhibition, ROS production, the expression of fibrogenic markers and hepatocyte apoptosis were reduced. NOX4 was expressed in human livers with stage 2–3 autoimmune hepatitis. Fibrosis was attenuated by the genetic deletion of NOX4. BDL mice gavaged with GKT137831 both in the preventive or the therapeutic arm displayed less ROS production; significantly attenuated fibrosis and decreased hepatocyte apoptosis. In conclusion, NOX4 plays a key role in liver fibrosis. GKT137831 is a potent inhibitor of fibrosis and hepatocyte apoptosis; therefore it is a promising therapeutic agent for future translational studies.