Drug-induced liver injury (DILI) is the leading cause of acute liver failure (ALF). Continuous and prolonged hepatic cellular oxidative stress and liver inflammatory stimuli are key signatures of DILI. DEAD-box helicase 3, X-linked (DDX3X) is a central regulator in pro-survival stress granule (SG) assembly in response to stress signals. However, the role of DDX3X in DILI remains unknown. Herein, we characterized the hepatocyte-specific role of DDX3X in DILI. Human liver tissues of DILI patients and control subjects were used to evaluate DDX3X expression. APAP, CCl4 and TAA models of DILI were established and compared between hepatocyte-specific DDX3X knockout (DDX3XΔhep) and wild-type control (DDX3Xfl/fl) mice. Hepatic expression of DDX3X was significantly decreased in the pathogenesis of DILI compared with controls in human and mice. Compared to DDX3Xfl/flmice, DDX3XΔhepmice developed significant liver injury in multiple DILI models. DDX3X deficiency aggravates APAP induced oxidative stress and hepatocyte death by affecting the pro-survival stress granule (SG) assembly. Moreover, DDX3X deficiency induces inflammatory responses and causes pronounced macrophage infiltration. The use of targeted DDX3X drug maybe promising for the treatment of DILI in human.