Obesity is a major health and economic burden. It is becoming increasingly common and is associated with serious and life threatening secondary complications, predominantly involving the cardiovascular system.Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases. Increased levels of plasma ADMA are associated with multiple pathologies; it is an established biomarker of cardiovascular disease and has been associated with obesity, type 2 diabetes, and insulin resistance. However at present little is understood about the direct effects of ADMA on lipid accumulation.Dimethylarginine dimethylaminohydrolase (DDAH) catalyses the metabolism of ADMA, there are two isoforms of the enzyme which are both involved in the control of ADMA and NO. In this study we show that hepatocyte specific knockout of ddah1 causes increased fat deposition.Adult hepatocyte specific ddah1 knockout mice and their wildtype counterparts were put on a 60% high fat diet for 12 weeks. On normal diet adult mice showed no difference in weight, fat mass or glucose handling and had no obvious liver dysfunction. Following high fat diet, hepatocyte ddah1 knockout mice have a significantly higher fat mass (wildtypes; 8.31 g ± 1.089, hepatocyte ddah1 knockout; 17.66 g ± 1.299) and overall weight (wildtypes; 41.47 g ± 1.788 hepatocyte ddah1 knockouts; 51.9 g ± 1.149). Glucose tolerance tests showed gross impairment in response to a bolus of glucose and fasting glucose levels were significantly different between wildtypes and knockouts (wildtypes; 7.133 mmol/l ± 0.333, hepatocyte ddah1 knockouts 9.20 mmol/l ± 0.5797). Indirect calorimetry was used to assess the metabolic rate of animals post high fat diet and showed hepatocyte specific ddah1 knockout animals to have a lower metabolic rate but no significant difference in activity or food intake. ADMA has been linked to increased mTOR gene expression and lipid biosynthesis in adipocytes. As a result we investigated gene expression in the liver of animals following high fat diet. Hepatocyte specific ddah1 knockouts showed significantly increased gene expression of both mTOR and SREBP1c. These data suggest that knockout of hepatic ddah1 and consequent increases in ADMA are novel regulators of lipid synthesis and weight gain on a high fat diet.