HDAC inhibitors induce histone hyperacetylation by a relative increase of histone acetyltransferase activity. Histone hyperacetylation may affect chromatin structure and susceptibility to DNA‐damaging stress, such as IR. We here investigate whether these inhibitors can radiosensitize human gastric MKN45 and colorectal DLD1 adenocarcinoma cells. In both cells, FK228 pretreatment at minimally toxic concentrations clearly augmented IR‐induced cell death, DNA fragmentation and caspase‐3/‐8 activation. In contrast, 5‐FU did not clearly augment IR‐induced cell death and caspase‐3 activation. FK228 increased expression of proapoptotic BH3‐only Bim proteins, and gene transfer–mediated overexpression of Bimα radiosensitized DLD1 cells. These data suggest that the FK228‐mediated increase of Bim expression may at least partially contribute to its augmentation of radiation‐induced apoptosis. However, FK228 did not distinctly affect IR‐induced phosphorylation of H2AX, which is an initial event followed by DNA damage. FK228 strongly augmented IR‐induced growth suppression of MKN45 tumor xenografts. In addition, other HDAC inhibitors, MS275 and CBHA, similarly augmented IR‐induced cell death in both cell types. Our results suggest that these HDAC inhibitors may enhance the efficacy of radiation therapy in gastrointestinal cancer cells. © 2004 Wiley‐Liss, Inc.