Identifying the vulnerability of altered DNA repair machinery that displays synthetic lethality withMYCNamplification is a therapeutic rationale in unfavourable neuroblastoma. However, none of the inhibitors for DNA repair proteins are established as standard therapy in neuroblastoma. Here, we investigated whether DNA-PK inhibitor (DNA-PKi) could inhibit the proliferation of spheroids derived from neuroblastomas of MYCN transgenic mice andMYCN-amplified neuroblastoma cell lines. DNA-PKi exhibited an inhibitory effect on the proliferation ofMYCN-driven neuroblastoma spheroids, whereas variable sensitivity was observed in those cell lines. Among them, the accelerated proliferation of IMR32 cells was dependent on DNA ligase 4 (LIG4), which comprises the canonical non-homologous end-joining pathway of DNA repair. Notably, LIG4 was identified as one of the worst prognostic factors in patients withMYCN-amplified neuroblastomas. It may play complementary roles in DNA-PK deficiency, suggesting the therapeutic potential of LIG4 inhibition in combination with DNA-PKi forMYCN-amplified neuroblastomas to overcome resistance to multimodal therapy.