Liver cancer susceptibility varies amongst humans and between experimental animal models because of multiple genetic and epigenetic factors. The molecular characterization of such susceptibilities has the potential to enhance cancer risk assessment of xenobiotic exposures and disease prevention strategies. Here, using DNase I hypersensitivity mapping coupled with transcriptomic profiling, we investigate perturbations in cis-acting gene regulatory elements associated with the early stages of phenobarbital (PB)-mediated liver tumor promotion in susceptible versus resistant mouse strains (B6C3F1 versus C57BL/6J). Integrated computational analyses of strain-selective changes in liver chromatin accessibility underlying PB response reveal differential epigenetic regulation of molecular pathways associated with PB-mediated tumor promotion, including Wnt/β-catenin signaling. Complementary transcription factor motif analyses reveal mouse strain-selective gene regulatory networks and a novel role for Stat, Smad, and Fox transcription factors in the early stages of PB-mediated tumor promotion. Mapping perturbations in cis-acting gene regulatory elements provides novel insights into the molecular basis for susceptibility to xenobiotic-induced rodent liver tumor promotion and has the potential to enhance mechanism-based cancer risk assessments of xenobiotic exposures.