Acute liver failure (ALF) has an abrupt onset with a frequently fatal outcome. Previous studies have found that oral antibiotics prevent drug-induced liver injury in animal experiments, indicating that the gut microbiota plays a critical role in the pathophysiological process. However, the underlying mechanism has not been fully understood. This study explored the comprehensive role of the gut microbiota in ALF using multi-omics. A cocktail of broad-spectrum antibiotics (Abx) pretreatment by gavage for four weeks improved the survival of D-(+)-galactosamine hydrochloride (D-Gal)/lipopolysaccharide (LPS)-induced ALF in C57BL/6 mice. RNA sequencing showed that inflammatory responses were inhibited and metabolic pathways were upregulated in the liver of Abx-treated ALF mice. The 16S rRNA gene sequencing revealed that Abx reshaped the composition and function of the gut microbiota, with an increased proportion of tryptophan (Trp) metabolism. In addition, global metabolic profiling by ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) indicated that the gut microbiota post-Abx intervention reduced Trp excretion, liberated more Trp to the host, and enhanced the kynurenine (Kyn) pathway with increased production of Kyn. As an endogenous aryl hydrocarbon receptor (AhR) ligand, Kyn has anti-inflammatory and immunosuppressive effects. Furthermore, AhR-targeted treatments affected the outcome of ALF mice with or without Abx pretreatment, indicating that AhR directly regulated susceptibility to ALF, at least in part. This study demonstrates that the gut microbiota-dependent control of the Trp metabolism could regulate host susceptibility to ALF by modulating the activity of AhR, and thus provides a promising target for better management of ALF.