Mammalian Hus1 plays an important role in maintaining genomic integrity. Cells lacking mouse Hus1 are hypersensitive to DNA damage inducers including UV and camptothecin (CPT). By using clonogenic assay, we show here that Hus1 deficient mouse cells are hypersensitive to ionizing radiation (IR) compared with their Hus1-positive counterparts. However, these cells show similar induction levels and similar rejoining rates of DNA double strand breaks (DSBs) following IR, indicating that the effect of Hus1 on cell radiosensitivity is independent of nonhomologous end-joining (NHEJ). By combining an I-SceI-induced-DNA DSBs system and a siRNA approach, we also show that knocking down Hus1 decreases the efficiency of homologous recombination repair (HRR), which is associated with the cellular sensitivity to IR-induced killing. Together, these results indicate that the role of Hus1 affecting the sensitivity of cells to IR-induced killing is independent of NHEJ but might be linked to HRR.