The gut–liver axis may provide a new perspective for treating anti-tuberculosis drug-induced liver injury (ATDILI). Herein, the protective effect ofLactobacillus casei(Lc) was investigated by modulating gut microflora (GM) and the toll like receptor 4 (TLR4)–nuclear factor (NF)-κB–myeloiddifferentiationfactor 88 (MyD88) pathway. C57BL/6J mice were given three levels of Lc intragastrically for 2 h before administering isoniazid and rifampicin for 8 weeks. Blood, liver, and colon tissues, as well as cecal contents, were collected for biochemical and histological examination, as well as Western blot, quantitative real time polymerase chain reaction (qRT-PCR), and 16S rRNA analyses. Lc intervention decreased alkaline phosphatase (ALP), superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), and tumor necrosis factor (TNF)-α levels (p< 0.05), recovered hepatic lobules, and reduced hepatocyte necrosis to alleviate liver injury induced by anti-tuberculosis drugs. Moreover, Lc also increased the abundance ofLactobacillusandDesulfovibrioand decreasedBilophilaabundance, while enhancing zona occludens (ZO)-1 and claudin-1 protein expression compared with the model group (p< 0.05). Furthermore, Lc pretreatment reduced the lipopolysaccharide (LPS) level and downregulated NF-κB and MyD88 protein expression (p< 0.05), thus restraining pathway activation. Spearman correlation analysis indicated thatLactobacillusandDesulfovibriowere positively correlated with ZO-1 or occludin protein expression and negatively correlated with pathway protein expression.Desulfovibriohad significant negative relationships with alanine aminotransferase (ALT) and LPS levels. In contrast,Bilophilahad negative associations with ZO-1, occludin, and claudin-1 protein expressions and positive correlations with LPS and pathway proteins. The results prove thatLactobacillus caseican enhance the intestinal barrier and change the composition of the gut microflora. Moreover,Lactobacillus caseimay also inhibit TLR4–NF-κB–MyD88 pathway activation and alleviate ATDILI.