Histone deacetylase (HDAC) inhibitors augment ionizing radiation (IR)-induced apoptosis in several cancer cells by undefined mechanism(s). We recently found that the HDAC inhibitors induce a BH3-only protein Bmf in human squamous carcinoma SAS cells. We extended this study and found that 2.5 nM FK228 pretreatment could not induce apoptosis but augmented IR-induced death. The FK228 pretreatment increased Bmf expression level, and siRNA-mediated knockdown of Bmf transcripts strongly inhibited its augmentation of IR-induced cell death, disruption of mitochondrial membrane potential and DNA fragmentation. Another HDAC inhibitor CBHA pretreatment similarly augmented IR-induced apoptosis, and this effect was also inhibited by Bmf knockdown. Bmf overexpression augmented IR-induced death, and the augmented effects of FK228 were similarly observed in another squamous carcinoma HSC2 cells. Overexpression of histone acetyltransferase p300 mimicked the effects of the HDAC inhibitors, i.e., it enhanced IR-induced death, which was mostly abolished by Bmf knockdown. Taken together, histone hyperacetylation may enhance IR-induced death via activation of Bmf transcription, thereby implying Bmf as a key molecule for HDAC inhibitors (FK228 and CBHA)-mediated enhancing effect on IR-induced cell death.