The Ets-1 transcription factor plays an important role in various physiological and pathological processes. These diverse roles of Ets-1 are likely to depend on its interaction partner proteins. We used our previously developed, recombinant biotinylated Ets-1 that conserves native Ets-1 properties to identify new interaction partners. Here, based on results from streptavidin pull-down assays, mass spectrometry and co-immunoprecipitation, we report a novel interaction partner for Ets-1 isoforms: a heterotrimeric complex of DNA-dependent protein kinase (DNA-PK), made up of Ku70, Ku86, and DNA-PK catalytic subunit (DNA-PKcs). Kinase assays performed in vitro showed that DNA-PK phosphorylates the Ets-1 protein. Furthermore, we demonstrated that Ku86, but not Ku70 or DNA-PKcs, down-regulated the transcriptional activity of Ets-1 when analysed using a reporter gene assay. These results illustrate how detecting novel molecular interactions may provide new clues for understanding the diverse functions of Ets-1.