The Ku heterodimer, a DNA repair protein complex consisting of 70- and 80-kDa subunits, is involved in the non-homologous end-joining (NHEJ) pathway. Plants are thought to use the NHEJ pathway primarily for the repair of DNA double-strand breaks (DSBs). The Ku70/80 protein has been identified in many plants and been shown to possess several similar functions to its counter protein complex in mammals. In the present study, ovate family protein 1 (AtOFP1) was demonstrated to be a plant Ku-interacting protein by yeast two-hybrid screening and the GST pull-down assay. Truncation analysis revealed that the C-terminal domain of AtKu70 contains interacting sites for AtOFP1. The electrophoretic mobility shift assay (EMSA) indicated that AtOFP1 is also a DNA binding protein with its binding domain at the N-terminus. In 3-week-old seedlings, expression of the AtOFP1 gene increased after exposure to DNA-damaging agents (such as methyl methanesulfonate (MMS) and menadione) in a time dependent manner. Seedlings lacking the AtOFP1 protein were more sensitive to MMS and menadione as compared with wild-type. Furthermore, similar to AtKu70(-/-) and AtKu80(-/-), the AtOFP1(-/-) mutant showed relatively lower NHEJ activity in vivo. Taken together, these results suggest that AtOFP1 may play a role in DNA repair through the NHEJ pathway accompanying with the AtKu protein.