The　emergence　of　chemoresistance　poses　a　significant　challenge　to　the　efficacy　of　DNA-damaging　agents　in　cancer　treatment,　in　part　due　to　the　inherent　DNA　repair　capabilities　of　cancer　cells.　The　Ku70/80　protein　complex　(Ku)　plays　a　central　role　in　double-strand　breaks　(DSBs)　repair　through　the　classical　non-homologous　end　joining　(c-NHEJ)　pathway,　and　has　proven　to　be　one　of　the　most　promising　drug　target　for　cancer　treatment　when　combined　with　radiotherapy　or　chemotherapy.　In　this　study,　we　conducted　a　high-throughput　screening　of　small-molecule　inhibitors　targeting　the　Ku　complex　by　using　a　fluorescence　polarization-based　DNA　binding　assay.　From　a　library　of　11,745　small　molecules,　UMI-77　was　identified　as　a　potent　Ku　inhibitor,　with　an　IC50　value　of　2.3　mu　M.　Surface　plasmon　resonance　and　molecular　docking　analyses　revealed　that　UMI-77　directly　bound　the　inner　side　of　Ku　ring,　thereby　disrupting　Ku　binding　with　DNA.　In　addition,　UMI-77　also　displayed　potent　inhibition　against　MUS81-EME1,　a　key　player　in　homologous　recombination　(HR),　demonstrating　its　potential　for　blocking　both　NHEJ-　and　HR-mediated　DSB　repair　pathways.　Further　cell-based　studies　showed　that　UMI-77　could　impair　bleomycin-induced　DNA　damage　repair,　and　significantly　sensitized　multiple　cancer　cell　lines　to　the　DNA-damaging　agents.　Finally,　in　a　mouse　xenograft　tumor　model,　UMI-77　significantly　enhanced　the　chemotherapeutic　efficacy　of　etoposide　with　little　adverse　physiological　effects.　Our　work　offers　a　new　avenue　to　combat　chemoresistance　in　cancer　treatment,　and　suggests　that　UMI-77　could　be　further　developed　as　a　promising　candidate　in　cancer　treatment.