Hepatocellular carcinoma (HCC) is a significant worldwide health concern that is associated with discrete etiological events, encompassing viral infection, metabolic stress and genotoxic compounds. In particular, exposure to the genotoxic hepatocarcinogen aflatoxin B1 (AFB1) is a significant factor in the genesis of human liver cancer. Presumably, genetic events associated with HCC could influence the effect of environmental insults, yielding a predilection for tumor development. The retinoblastoma (RB) tumor suppressor pathway is functionally inactivated in HCC through discrete mechanisms; however, the role of RB in suppressing tumorigenesis in this disease is poorly understood. Therefore, we analysed how RB status affects the response to AFB1 in reference to acute exposures and tumor development reflective of chronic exposure. Liver-specific Rb deletion resulted in an aberrant proliferative response to AFB1. This cell-cycle induction was associated with increased levels of secondary genetic damage and failure in appropriate cell-cycle coupling. This effect of RB loss was unique to AFB1 and involved the induction of a non-canonical proliferative pathway, and was not merely reflective of the overall cell-cycle deregulation or aberrant regenerative responses. The acute responses to AFB1 exposure presaged aberrations in hepatocyte nuclear morphology and ploidy with RB loss. Correspondingly, RB-deficient livers showed significantly enhanced susceptibility to liver tumorigenesis initiated by AFB1. Combined, these studies show that RB has a critical role in mediating checkpoint responses in liver tissue to maintain genome integrity and in suppressing tumorigenesis.