Class IIa histone deacetylases (HDACs) are signal-responsive regulators of gene expression involved in vascular homeostasis. To investigate the differential role of class IIa HDACs for the regulation of angiogenesis, we used siRNA to specifically suppress the individual HDAC isoenzymes. Silencing of HDAC5 exhibited a unique pro-angiogenic effect evidenced by increased endothelial cell migration, sprouting, and tube formation. Consistently, overexpression of HDAC5 decreased sprout formation, indicating that HDAC5 is a negative regulator of angiogenesis. The antiangiogenic activity of HDAC5 was independent of myocyte enhancer factor-2 binding and its deacetylase activity but required a nuclear localization indicating that HDAC5 might affect the transcriptional regulation of gene expression. To identify putative HDAC5 targets, we performed microarray expression analysis. Silencing of HDAC5 increased the expression of fibroblast growth factor 2 (FGF2) and angiogenic guidance factors, including Slit2. Antagonization of FGF2 or Slit2 reduced sprout induction in response to HDAC5 siRNA. Chromatin immunoprecipitation assays demonstrate that HDAC5 binds to the promoter of FGF2 and Slit2. In summary, HDAC5 represses angiogenic genes, such as FGF2 and Slit2, which causally contribute to capillary-like sprouting of endothelial cells. The derepression of angiogenic genes by HDAC5 inactivation may provide a useful therapeutic target for induction of angiogenesis.