AbstractHistone deacetylases (HDACs) modulate the transcription of a subset of genes by various means. HDAC5 is a class II HDAC whose subcellular location is signal‐dependent. At present, its known gene targets are few in number. Here we identify a new HDAC5 target: the gene encoding the cell cycle‐regulatory protein cyclin D3. When overexpressed in Balb/c‐3T3 cells or mouse embryo fibroblasts, HDAC5 substantially reduced the activity of the cyclin D3 promoter and the abundance of endogenous cyclin D3 protein. Conversely, conditions that blocked HDAC5 function increased cyclin D3 expression: treatment of cells with the class I/II HDAC inhibitor trichostatin A (TSA), depletion of HDAC5 from cells by RNA interference, and cytoplasmic sequestration of HDAC5 by co‐expression of catalytically active calcium/calmodulin‐dependent protein kinase. HDAC5 interacted with the cyclin D3 promoter in vivo, and the HDAC5‐responsive element was within 118 base pairs upstream of the transcription start site. Mutation of the Sp1 site and the cyclic AMP response element within this region did not affect the responsiveness of the cyclin D3 promoter to HDAC5 or TSA. J. Cell. Biochem. 104: 2143–2154, 2008. © 2008 Wiley‐Liss, Inc.