Hepatoblastoma (HB) is the most common malignant liver tumor in children. DNA and DNA-associated processes are one of the most important targets of chemotherapeutic agents. Isoorientin (Iso), a natural flavonoid compound, can be extracted from several plant species. The effects of Iso and its molecular mechanisms on hepatic malignancies remain unclear. Herein, the anti-tumor effects of Iso in HB and its underlying mechanisms were explored. We found that Iso significantly inhibited the proliferation of HB cells both in vitro and in vivo. Mechanistic studies showed that Iso triggered cell apoptosis by inducing DNA double-stranded breaks and blocking the initiation process of homologous recombination repair, which was related to the attenuation of ataxia telangiectasia mutated (ATM) activation and inhibiting the binding of phosphorylated ataxia telangiectasia mutated (pATM) and the MRE11-RAD50-NBS1 (MRN) complex. Furthermore, Iso markedly sensitized HB cells to the anti-proliferative effects of the poly ADP-ribose polymerase (PARP) inhibitor olaparib both in vivo and in vitro. Taken together, our study first showed that Iso was a DNA-damage agent, and the combination of Iso with a PARP inhibitor might be a promising strategy for treating HB patients.