AbstractRadiotherapy can induce various adverse effects including fibrosis in cancer patients. Radiation‐induced aberrant expression of profibrotic genes has been associated with dysregulated epigenetic mechanisms. Pan‐BET (bromodomain and extraterminal domain) inhibitors, such as JQ1 and I‐BET151, have been reported to attenuate the profibrotic response after irradiation. Despite their profound preclinical efficacy, the clinical utility of pan‐inhibitors is limited due to observed cytotoxicicities. Recently, inhibitors were developed that selectively target the first (BD1) and second (BD2) bromodomain of the BET proteins (iBET‐BD1 [GSK778] and iBET‐BD2 [GSK046]). Here, their potential to attenuate radiation‐induced fibroblast activation with low‐toxicity was investigated. Our results indicated that cell proliferation and cell cycle progression in fibroblasts from BJ cells and six donors were reduced when treated with I‐BET151 and iBET‐BD1, but not with iBET‐BD2. After irradiation, induction of DGKA and profibrotic markers, especially COL1A1 and ACTA2, was attenuated with all BET inhibitors. H3K27ac enrichment was similar at the DGKA enhancer region after I‐BET151 treatment and irradiation, but was reduced at the COL1A1 transcription start site and the ACTA2 enhancer site. iBET‐BD2 did not change H3K27ac levels in these regions. BRD4 occupancy at these regions was not altered by any of the compounds. Cell migration activity was measured as a characteristic independent of extracellular matrix production and was unchanged in fibroblasts after irradiation and BET inhibitor‐treatment. In conclusion, iBET‐BD2 efficiently suppressed radiation‐induced expression of DGKA and profibrotic markers without showing cytotoxicity. Thus BD2‐selective targeting is a promising new therapeutic avenue for further investigations to prevent or attenuate radiotherapy‐induced fibrosis.