The lysine acetylation state of nonhistone proteins may be regulated through histone deacetylases (HDACs). Evidence suggests that nitric oxide (NO) synthase 3 (NOS3; endothelial NOS) is posttranslationally lysine acetylated, leading to increased NO production in the endothelium. We tested the hypothesis that NOS3 is lysine acetylated and that upregulated HDAC1-mediated deacetylation leads to reduced NO production in endothelial cells. We determined that NOS3 is basally lysine acetylated in cultured bovine aortic endothelial cells (BAECs). In BAECs, HDAC1 is expressed in the nucleus and cytosol and forms a novel protein-protein interaction with NOS3. Overexpression of HDAC1 in BAECs resulted in a significant reduction in NOS3 lysine acetylation (control = 1.0 ± 0.1 and HDAC1 = 0.59 ± 0.08 arbitrary units, P < 0.01) and significantly blunted basal nitrite production (control 287.7 ± 29.1 and HDAC1 172.4 ± 31.7 pmol·mg-1·h-1, P < 0.05) as well as attenuating endothelin-1-stimulated nitrite production (control = 481.8 ± 50.3 and HDAC1 243.1 ± 48.2 pmol·mg-1·h-1, P < 0.05). While HDAC1 knockdown with small-interfering RNA resulted in no change in NOS3 acetylation level, yet increased basal nitrite production (730.6 ± 99.1 pmol·mg-1·h-1) and further exaggerated increases in endothelin-1 stimulated nitrite production (1276.9 ± 288.2 pmol·mg-1·h-1) was observed. Moreover, overexpression or knockdown of HDAC1 resulted in no significant effect on NOS3 protein expression or NOS3 phosphorylation sites T497, S635, or S1179. Thus these data indicate that upregulated HDAC1 decreases NOS3 activity, most likely through direct lysine deacetylation of NOS3. We propose that HDAC1-mediated deacetylation of NOS3 may represent a novel target for endothelial dysfunction. © 2014 the American Physiological Society.