Acetaminophen (APAP) overdose is a leading cause of acute liver failure (ALF), primarily through the excessive production of N-acetyl-p-benzoquinone imine (NAPQI). N-acetylcysteine (NAC) is the FDA-approved treatment for APAP overdose, but there is a growing interest in microRNAs as potential therapeutic agents. We delivered miR-375 ectopically via a liver-tropic adeno-associated virus serotype 8 (AAV8) and demonstrated its potent protection in a murine model of APAP overdose-induced ALF. Slc16a2, Cyb5b, and Acsl5 were identified as critical targets acting synergistically to mitigate toxicity. Liver transcriptome revealed that miR-375 overexpression or silencing of the targets of miR-375 increased Gstm3 expression in mice. AAV8-mediated Gstm3 expression protects against APAP-ALF, and the protection was further enhanced by disrupting the expression of Cyp2e1. Additionally, CYP2E1 and GSS, which contribute to APAP detoxification, were down- and upregulated by miR-375, respectively. These findings suggest that miR-375 prevents APAP-ALF by orchestrating the expression of pharmacogenes and enhancing glutathione synthesis. We conclude that miR-375 and its targets are promising therapeutic targets for APAP-ALF.