NCX 4016 is a nitric oxide (NO)-donating derivative of acetylsalicylic acid. NO and salicylate, in vivo metabolites of NCX 4016, were shown to be potential actors in controlling glucose homeostasis. In this study, we evaluated the action of NCX 4016 on the capacity of 3T3-L1 adipocytes to transport glucose in basal and insulin-stimulated conditions. NCX 4016 induced a two fold increase in glucose uptake and the translocation of the glucose transporters GLUT1 and GLUT4 to the plasma membrane, leaving unaffected their expression. Importantly, NCX 4016 further increased glucose transport induced by a physiological concentration of insulin. This stimulatory effect of NCX 4016 appears to be mediated by its NO moiety. Indeed, it is inhibited by a NO scavenger and treatment with acetylsalicylic or salicylic acid had no effect. Although NO is involved in the action of NCX 4016, it did not mainly depend on the soluble cGMP-cyclase/protein kinase G pathway. Furthermore, NCX 4016-stimulated glucose transport did not involve the insulin signaling cascade required to stimulate glucose transport. NCX 4016 induces a small activation of the MAP kinases p38 and JNK, and no activation of other stress-activated signaling molecules including ERK, IB, or AMP-activated kinases. Interestingly, NCX 4016 modified the content of S-nitrosylated proteins in adipocytes. Taken together, our results indicate that NCX 4016 induced glucose transport in adipocyte through a novel mechanism possibly involving S-nitrosylation. NCX 4016 thus possesses interesting characteristics to be considered as a candidate molecule for treatment of patients suffering from metabolic syndrome and type 2 diabetes.