Introduction: Nitric oxide (NO) is a key regulator of endothelial cell function and has been shown to affect cell motility via Rho GTPases, key regulators of actin dynamics. However, its role in immune cells has not been studied in detail. Macrophages and oxy- low density lipoprotein are the predominant cause of atheroma, which leads to atherosclerosis. If NO controls macrophage motility as it does endothelial cell motility, then the development of atheroma may modulated by the addition of specific NO inhibitors. Methods: Primary peritoneal macrophages were obtained from wild type (WT) and dimethylarginine dimethylaminohydrolase 2 macrophage specific knock-out (DDAH2 mac-/-) mice. The knockout model was verified using flow cytometry for the macrophage specific marker F4-80. Their cell motility was tracked for 20 hours following incubation with; NO inhibitor asymmetric dimethylarginine (ADMA), non-NO inhibitor symmetric dimethylarginine (SDMA), NO donor in control conditions or with an inflammatory cytokine treatment. Results were analysed manually using an Image J (Licor) tracking program. Nitrite levels and cell phagocytosis were determined using the Griess assay and the measurement of an E-coli fluorescent Bio-particle ingested by macrophages. Immunohistochemistry of F-actin using a phalloidin stain was used to investigate the effect of the NO/ADMA pathway on GTPases in particular stress fibre formation which controls retraction of cell end and directional movement. Results: DDAH2 mac-/- macrophages moved significantly slower and less far than WT macrophages in control conditions (0.252±0.0182 µm/min; 0.151±0.0290 µm/min, for WT and DDAH2 mac-/- respectively where p<0.05 and n=45cells from 3 mice). The addition of an NO donor significantly increased motility in DDAH2 mac-/- (0.213±0.0198 µm/min where p<0.05 and n=45 cells from 3 mice). Inflammatory cocktail-treated macrophages from DDAH2 mac-/- had significantly lower nitrite levels over a total period of 48 hours (42.63±4.79 µmol; 22.05±4.50 µmol for WT and DDAH2 mac-/- respectively where p<0.05 and n=8 WT and n=9 DDAH2 mac-/-) and less phagocytosis when treated with an inflammatory cocktail (5.346±2.723 au; 2.335±1.338 au, for WT and DDAH2 mac-/- respectively where P=NS and n=4). F-actin staining revealed more stress fibres around the periphery of cells and the intracellular in DDAH2 mac-/- animals when compared to WT. Conclusion: NO reduction in macrophages, via the deletion of DDAH2, leads to reduced motility, nitrite production and phagocytosis indicating that NO is critical for the function of these immune cells. Increased stress fibres with DDAH2 mac-/- highlights that this effect is mediated by GTPases. The reduction in macrophage motility and phagocytosis capability suggests a possible therapeutic against the development of plaque formation in atherosclerosis.