Epithelial to mesenchymal transition is a common event during tumour dissemination. However, direct epithelial to amoeboid transition has not been characterized to date. Here we provide evidence that cells from hepatocellular carcinoma (HCC), a highly metastatic cancer, undergo epithelial to amoeboid transition in physiological environments, such as organoids or threedimensional complex matrices. Furthermore, the NADPH oxidase NOX4 inhibits this transition and therefore suppresses efficient amoeboid bleb-based invasion. Moreover, NOX4 expression is associated with E-cadherin levels and inversely correlated with invasive features. NOX4 is necessary to maintain parenchymal structures, increase cell–cell and cell-to-matrix adhesion, and impair actomyosin contractility and amoeboid invasion. Importantly, NOX4 gene deletions are frequent in HCC patients, correlating with higher tumour grade. Contrary to that observed in mesenchymal cell types, here NOX4 suppresses Rho and Cdc42 GTPase expression and downstream actomyosin contractility. In HCC patients, NOX4 expression inversely correlates with RhoC and Cdc42 levels. Moreover, low expression of NOX4 combined with high expression of either RhoC or Cdc42 is associated with worse prognosis. Therefore, loss of NOX4 increases actomyosin levels and favours an epithelial to amoeboid transition contributing to tumour aggressiveness.