Histone deacetylase (HDAC) inhibitors modify transcription of selected genes and eventually induce apoptosis. However, molecular mechanisms for their proapoptotic activity remain unclear. We here demonstrate that HDAC inhibitors FK228 and CBHA preferentially upregulated the BH3-only protein Bmf in a broad range of cancer cells. In contrast, HDAC1 overexpression distinctly reduced Bmf expression. FK228 induced histones H3 and H4 acetylation at Bmf promoter region, but not at its 3′ region, suggesting that histone hyperacetylation causes Bmf transcriptional activation. Knockdown of Bmf transcripts rescued cells from FK228 or CBHA-induced cell death, disruption of mitochondrial membrane potential (ΔΨm) and DNA fragmentation. Taken together, FK228 and CBHA activate Bmf transcription by histone hyperacetylation at its promoter region, and inhibition of this action decreased their proapoptotic activity, thereby highlighting a central role of Bmf in HDAC inhibitor-mediated apoptosis.