Tuberculosis (TB) is one of the most prevalent infections. However, anti-TB drugs induce adverse liver injury in up to 40% of patients. Studies on candidate genes have suggested that single-nucleotide polymorphisms account for only a small contribution to the occurrence of anti-TB drug-induced liver injury (ATLI). In this study, whole-genome DNA methylation analysis was performed to systematically screen the ATLI-associated factors in a 49 vs. 51 case-control population. Next, 34 identified candidate probes were validated using MassARRAY in 296 cases and 288 controls. Our results indicated that 12 CpG sites on seven probes were positively associated with ATLI risk. Furthermore, we applied a CRISPR/Cas9-mediated methylation modifiable cell model and demonstrated that four CpGs in or near the gene region of AK2, SLC8A2, and PSTPIP2 affected the cellular response to rifampicin treatment. This study provides new biomarkers associated with ATLI occurrence.Tuberculosis (TB) is one of the most prevalent chronic bacterial infections, with >6 million new cases occurring globally each year. 1 Although the first-line therapy with isoniazid, rifampicin, pyrazinamide, and ethambutol is able to suppress the increasing incidence of TB, 1 it has been reported that ~ 0.8-40% of patients worldwide suffer from anti-TB drug-induced liver injury (ATLI). 2 ATLI is the most common side effect of anti-TB drugs and often leads to the delayed completion or failure of chemotherapy in patients. 3,4 In some extreme cases, ATLI causes irreversible hepatonecrosis or death in patients receiving anti-TB treatment.Recently, the mortality rate of ATLI was estimated to be as high as 22.7%. 2 Therefore, ATLI is regarded as a severe clinical problem that poses a significant challenge to the early control of TB progression.Numerous association studies have attempted to identify the risk factors for ATLI. A number of genetic single-nucleotide polymorphisms (SNPs) in various genes have been reported to be related to the development of ATLI. The majority of these identified genes are involved in the pathways of drug absorption, distribution, metabolism, and excretion (ADME), acquired immunity, innate immune