© 2016 the American Physiological Society. Nitric oxide synthase 1 (NOS1)- derived nitric oxide (NO) production in collecting ducts is critical for maintaining fluid-electrolyte balance. Rat collecting ducts express both the full-length NOS1α and its truncated variant NOS1β, while NOS1β predominates in mouse collecting ducts. We reported that dynamin-2 (DNM2), a protein involved in excising vesicles from the plasma membrane, and NOS1α form a protein-protein interaction that promotes NO production in rat collecting ducts. NOS1β was found to be highly expressed in human renal cortical/medullary samples; hence, we tested the hypothesis that DNM2 is a positive regulator of NOS1β-derived NO production. COS7 and mouse inner medullary collecting duct-3 (mIMCD3) cells were transfected with NOS1β and/or DNM2. Coimmunoprecipitation experiments show that NOS1β and DNM2 formed a protein-protein interaction. DNM2 overexpression decreased nitrite production (index of NO) in both COS7 and mIMCD-3 cells by 50–75%. mIMCD-3 cells treated with a panel of dynamin inhibitors or DNM2 siRNA displayed increased nitrite production. To elucidate the physiological significance of IMCD DNM2/NOS1β regulation in vivo, flox control and CDNOS1 knockout mice were placed on a high-salt diet, and freshly isolated IMCDs were treated acutely with a dynamin inhibitor. Dynamin inhibition increased nitrite production by IMCDs from flox mice. This response was blunted (but not abolished) in collecting duct-specific NOS1 knockout mice, suggesting that DNM2 also negatively regulates NOS3 in the mouse IMCD. We conclude that DNM2 is a novel negative regulator of NO production in mouse collecting ducts. We propose that DNM2 acts as a “break” to prevent excess or potentially toxic NO levels under high-salt conditions.