Acetaminophen toxicity is a leading cause of acute liver failure (ALF). We found that miRNA-122 (miR-122) is down-regulated in liver biopsy specimens of patients with ALF and in acetaminophen-treated mice. A marked decrease in the primary miR-122 expression occurs in mice on acetaminophen overdose because of suppression of its key transactivators, hepatocyte nuclear factor (HNF)-4α and HNF6. More importantly, the mortality rates of male and female liver-specific miR-122 knockout (LKO) mice were significantly higher than control mice when injected i.p. with an acetaminophen dose not lethal to the control. LKO livers exhibited higher basal expression of cytochrome P450 family 2 subfamily E member 1 (CYP2E1) and cytochrome P450 family 1 subfamily A member 2 (CYP1A2) that convert acetaminophen to highly reactive N-acetyl-p-benzoquinone imine. Upregulation of Cyp1a2 primary transcript and mRNA in LKO mice correlated with the elevation of aryl hydrocarbon receptor (AHR) and mediator 1 (MED1), two transactivators of Cyp1a2. Analysis of ChIP-seq data in the ENCODE (Encyclopedia of DNA Element) database identified association of CCCTC-binding factor (CTCF) with Ahr promoter in mouse livers. Both MED1 and CTCF are validated conserved miR-122 targets. Furthermore, depletion of Ahr, Med1, or Ctcf in Mir122^−/− hepatocytes reduced Cyp1a2 expression. Pulse-chase studies found that CYP2E1 protein level is upregulated in LKO hepatocytes. Notably, miR-122 depletion sensitized differentiated human HepaRG cells to acetaminophen toxicity that correlated with upregulation of AHR, MED1, and CYP1A2 expression. Collectively, these results reveal a critical role of miR-122 in acetaminophen detoxification and implicate its therapeutic potential in patients with ALF.