Metabolic syndrome is a major healthcare and economic burden. Cardiovascular disease and obesity are major symptoms of metabolic syndrome. Asymmetric dimethylarginine (ADMA) is an endogenously produced inhibitor of nitric oxide synthesis that is an established biomarker of cardiovascular disease. Increased plasma concentrations of ADMA have been reported in obese individuals in whom it is thought to contribute to cardiovascular dysfunction. However, at present little is known about the direct effects of ADMA on adipocytes and obesity. In the present study we examined the effects of ADMA on 3T3-L1 cells in vitro and on adipose tissue in vivo. Mouse 3T3-L1 cells were fully differentiated to adipocytes and treated with exogenous ADMA (10μM) after which intracellular neutral lipid was stained with oil red O and adipocyte area quantified by microscopy. To specifically elevate ADMA concentrations in adipocytes in vivo we deleted the enzyme dimethylarginine dimethylaminohydrolase 1 (DDAH1, the predominant enzyme responsible for intracellular ADMA metabolism) in adipocytes using adiponectin driven Cre expression1. In vitro treatment with ADMA caused adipocyte hypertrophy (control area 27.65μM2 ± 1.41, 10µM ADMA area 33.85μM2 ± 1.62, p>0.01, n=6, all results ±SEM and student T test ) which was independent of NO blockade as the nitric oxide synthase inhibitors L-NAME (1mM) and PB-ITU (20μM) had no effect on lipid accumulation (28.02μM2 ± 1.52 and 25.73μM2 ± 1.27 respectively). ADMA treatment of 3T3-L1 cells (10μM, 48h) resulted in increased expression of mTOR RNA and protein (2.4X ±0.47, and 1.73X ±0.22 respectively, p>0.05, n=3). Downstream of mTOR the targets of the transcription factor SREBP1c including ACC and FASN were upregulated (4.6±0.8 and 1.4±0.16 fold respectively, p>0.05, n=6) suggesting that ADMA increases adipocyte size through the upregulation of lipid biosynthesis in an NO independent manner. Adipocytes isolated from AdCre-DDAH1-/- mice were found to have increased intracellular ADMA (control 72.1 ±22.36 nmol/mg, KO 85.24 ±11.90 nmol/mg) which caused a corresponding decrease in intracellular NO concentration (control 30.66µM/mg ± 4.9, KO 18.22µM/mg ± 3.86, p>0.05, n=4). Immunohistochemistry revealed that adipocytes within the epididymal depot were larger in AdCre-DDAH1-/- mice than wildtype littermates (18.05±1.01µM2 and 14.47±0.52µM2 respectively, p>0.05, n=4). The physiological consequence of this adipocyte hypertrophy was increased macrophage infiltration (control 17.9±0.9, KO 22.2±1.2 cells per frame, p<0.05, n=4). These data suggest that ADMA is a novel regulator of lipid biosynthesis within adipocytes independent to its actions as a nitric oxide synthase inhibitor. Adipocyte hypertrophy causes increased inflammation and is likely to contribute to insulin resistance.