Non-homologous end joining (NHEJ) is a pivotal mechanism in the repair of DNA double-strand breaks (DSBs). Central to NHEJ is the DNA-dependent protein kinase (DNA-PK) complex, comprising the KU heterodimer and the catalytic subunit, DNA-PKcs. In this study, we characterize TOX High Mobility Group Box Family Member 4 (TOX4) as a factor recruited to both laser-induced DNA damage and endonuclease-induced DNA DSBs. Depletion of TOX4 leads to accumulation of DNA damage, which is epistatic to DNA-PKcs. Consistently, TOX4 depletion substantially reduces NHEJ efficiency measured using both intrachromosomal and extrachromosomal repair assays. Our proteomic and biochemical analyses reveal TOX4 association with DNA-PK that is required for DNA-PKcs activation. Furthermore, we show that TOX4 coordinates with Phosphatase 1 Nuclear-Targeting Subunit (PNUTS) in NHEJ. PNUTS, previously shown to protect DNA-PKcs phosphorylation from protein phosphatase 1 (PP1)-mediated dephosphorylation, binds DNA-PK in a TOX4-dependent manner. In line with its role in DNA repair, TOX4 emerges as a promising target for anti-cancer drug development, and its targeting enhances tumor cell sensitivity to DNA damage in head and neck cancer and other malignancies.