Updated project metadata. Hepatocyte nuclear factor 4 (HNF4) is a transcription factor that acts as a master regulator of genes in several endodermal-derived tissues, including the intestine in which it plays a central role during development and tumorigenesis. To better delineate the mechanisms by which HNF4 can interfere during these processes, we combined stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein (GFP) as well as with proximity-dependent purification by the biotin ligase BirA, both fused to HNF4. Surprisingly, these analyses identified a significant enrichment of proteins falling into the DNA repair gene ontology term, a so far unidentified biological feature of this transcription factor. Several of these proteins including PARP1, RAD50 and DNA-PKcs were confirmed to interact with HNF4in colorectal cancer cell lines. During DNA damage response, HNF4 localized to double strand DNA breaks in these cells. HNF4was able to interfere functionally during non-homologous end-joining (NHEJ). Overall, these observations identify an unsuspected role for this transcription factor during the DNA damage response.