OBJECTIVES: Drug-induced liver injury (DILI) can be improved by modulating gut microbiota. We aimed to investigate a probiotic mixture comprising Bifidobacterium Longum, Streptococcus thermophilus, and Lactobacillus delbrueckii subspecies bulgaricus (BSL) in mitigating acetaminophen induced liver injury (AILI), and to elucidate the underlying mechanisms. METHODS: Gut bacterial communities were analyzed in fecal samples from patients with DILI and healthy controls. Mice were pretreated with BSL or PBS for 10 days, then subjected to a single dose of acetaminophen (300 mg/kg) gavage and euthanized 24 h later. Transcriptome sequencing, microbiome, and metabolome sequencing were performed on mouse samples, respectively. RESULTS: Gut bacterial dysbiosis existed in DILI patients, with a decrease in Gram-positive bacteria and an increase in Gram-negative bacteria. A similar situation occurred in AILI mice. Pretreatment of BSL significantly improved APAP-induced disorders of gut bacteria and alleviated hepatic inflammation and necrosis. Transcriptome sequencing showed that BSL inhibited the hepatic damage pathways, such as Hippo and TGF-β signaling pathway. Metabolomic profiling revealed an obvious increase in oligopeptides containing branched-chain amino acids (BCAAs) in AILI mice, whereas these metabolites were significantly negatively correlated with the abundance of BSL, but positively with key genes of Hippo pathway. In vitro experiments showed that leucine exerted a dose-related exacerbation pattern on APAP-mediated hepatocellular injury. Mice supplemented with leucine resulted in the further overexpression of Yes-associated protein, an increase in oxidative stress, and a worsening of AILI.