There is evidence that nitric oxide (NO) is required for the normal increases in skeletal muscle glucose uptake during contraction, but the mechanisms involved have not been elucidated. We examined whether NO regulates glucose uptake during skeletal muscle contractions via cGMP-dependent or cGMP-independent pathways. Isolated extensor digitorum longus (EDL) muscles from mice were stimulated to contract ex vivo, and potential NO signaling pathways were blocked by the addition of inhibitors to the incubation medium. Contraction increased (P < 0.05) NO synthase (NOS) activity (∼40%) and dichlorofluorescein (DCF) fluorescence (a marker of oxidant levels; ∼95%), which was prevented with a NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA), and antioxidants [nonspecific antioxidant, N-acetylcysteine (NAC); thiol-reducing agent, DTT], respectively. L-NMMA and NAC both attenuated glucose uptake during contraction by ∼50% (P < 0.05), and their effects were not additive. Neither the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, which prevents the formation of cGMP, the cGMP-dependent protein (PKG) inhibitor Rp-8-bromo-β-phenyl-1,N2-ethenoguanosine 3',5'-cyclic monophosphorothioate sodium salt nor white light, which breaks S-nitrosylated bonds, affects glucose uptake during contraction; however, DTT attenuated (P < 0.05) contraction-stimulated glucose uptake (by 70%). NOS inhibition and antioxidant treatment reduced contraction-stimulated increases in protein S-glutathionylation and tyrosine nitration (P < 0.05), without affecting AMPK or p38 MAPK phosphorylation. In conclusion, we provide evidence to suggest that NOS-derived oxidants regulate skeletal muscle glucose uptake during ex vivo contractions via a cGMP/PKG-, AMPK-, and p38 MAPK-independent pathway. In addition, it appears that NO and ROS may regulate skeletal muscle glucose uptake during contraction through a similar pathway.