The role of dimethylarginine dimethylaminohydrolase 2 (DDAH2) in glucose metabolism is unknown. Here, we generated DDAH2 transgenic (Tg) mice. These mice had lower plasma glucose levels (60 min: 298±32 vs. 418±35 mg/dl; 120 min: 205±15 vs. 284±20 mg/dl) and higher insulin levels (15 min: 2.1±0.2 vs. 1.5±0.1 ng/ml; 30 min: 1.8±0.1 vs. 1.5±0.1 ng/ml) during intraperitoneal glucose tolerance tests when fed a high‐fat diet (HFD) compared with HFD‐fed wild‐type (WT) mice. Glucose‐stimulated insulin secretion (GSIS) was increased in Tg islets by 33%. Pancreatic asymmetrical dimethylarginine, nitric oxide, and oxidative stress levels were not correlated with improvements in insulin secretion in Tg mice. Secretagogin, an insulin vesicle docking protein, was up‐regulated by 2.7‐fold in Tg mice and in pancreatic MIN‐6 cells overexpressing DDAH2. GSIS in MIN‐6 cells was dependent on DDAH2‐induced secretagogin expression. Pancreatic Sirt1, DDAH2, and secretagogin were down‐regulated in HFD‐fed WT mice by 70, 75, and 85%, respectively. Overexpression of Sirt1 overexpression by 3.9‐fold increased DDAH2 and secretagogin expression in MIN‐6 cells by 3.2‐ and 2.5‐fold, respectively. DDAH2 overexpression improved GSIS in pancreas‐specific Sirt1‐deficient mice. In summary, the Sirt1/DDAH2/secretagogin pathway is a novel regulator of GSIS.—Hasegawa, K, Wakino, S., Kimoto, M, Minakuchi, H., Fujimura, K., Hosoya, K., Komatsu, M., Kaneko, Y., Kanda, T., Tokuyama, H., Hayashi, K., Itoh, H. The hydrolase DDAH2 enhances pancreatic insulin secretion by transcriptional regulation of secretagogin through a Sirt1‐dependent mechanism in mice. FASEB J. 27, 2301–2315 (2013). www.fasebj.org