XRCC4 is one of the core factors for DNA double-strand break (DSB) repair through non-homologous end joining (NHEJ). XRCC4 is phosphorylated by DNA-dependent protein kinase (DNA-PK), with Ser260 and Ser320 (Ser318 in the alternatively spliced form) being the major phosphorylation sites in vitro. It was recently reported that Ser320 is phosphorylated by DNA-PK in response to DNA damage; however, it is currently unclear whether Ser260 is phosphorylated in cellulo in response to DNA damage. Herein, we generated an antibody against XRCC4 phosphorylated on Ser260 and examined its phosphorylation status via Western blotting. XRCC4 Ser260 phosphorylation increased after irradiation with 30-300 Gy of γ-rays and was suppressed by DNA-PK inhibitor but not by ATM inhibitor. Moreover, XRCC4 Ser260 phosphorylation decreased in DNA-PKcs-deficient cells. These observations indicate that XRCC4 Ser260 is phosphorylated by DNA-PK in cellulo. The XRCC4S260A mutant reversed the high radiosensitivity of XRCC4-deficient M10 cells to a similar level to that of wild-type XRCC4. However, the clonogenic survival of cells expressing the XRCC4S260A mutant was slightly but significantly lower than that of those expressing wild-type XRCC4. In addition, XRCC4S260A-expressing cells displayed a significantly greater number of γ-H2AX foci than XRCC4WT-expressing cells 4 h after 1 Gy irradiation and without irradiation. The present results suggest a potential role of XRCC4 Ser260 phosphorylation by DNA-PK in DSB repair.