Contraction-stimulated skeletal muscle glucose uptake is distinct from insulin and is an effective alternative for the regulation of blood glucose levels in diabetic subjects. Regulation of muscle glucose uptake during contraction/exercise is complex with likely redundancy involving AMPK, CaMK, PKC, reactive oxygen species and nitric oxide (NO). In this study, we investigated the role of neuronal nitric oxide synthase mu (nNOSμ) in muscle glucose uptake during contraction using nNOSμ knockout and wild type mice. EDL muscles were isolated and contracted ex vivo and glucose uptake was determined during contraction using dual tracers. Body mass (28.6±0.7 versus 26.3±0.6 g, P<0.001) and EDL mass (10.9±0.2 versus 9.9±0.3 mg, P<0.01) were lower in nNOSμ knockout mice. Muscle glucose uptake at rest (knockout: 0.63±0.04; wild type: 0.52±0.04 µmol/g/hr, P>0.05) and during contraction (knockout: 2.06±0.07; wild type: 2.02±0.07 µmol/g/hr, P>0.05) were normal in nNOSμ knockout mice. However, N-monomethyl L-arginine (L-NMMA) (a non-specific NOS competitive inhibitor) attenuated (P<0.001) the increase in contraction-stimulated glucose uptake to a similar extent in both genotypes (knockout: 1.71±0.05; wild type: 1.72±0.06 µmol/g/hr). The NOS substrate, L-arginine, overcame the L-NMMA attenuation of glucose uptake during contraction. No difference in contraction force per muscle mass was observed between genotypes and with L-NMMA or L-arginine incubation. This indicated that the attenuation of glucose uptake during contraction in both genotypes was due to a direct specific effect of L-NMMA on NOS. Mouse skeletal muscle also expresses the nNOS splice variant, nNOSβ, which is not affected in nNOSμ knockout mice. Neuronal NOSβ is important for cytoskeleton integrity and cytoskeletal re-arrangement appears critical for contraction-stimulated skeletal muscle glucose uptake. A similar effect of L-NMMA on glucose uptake during contraction in both nNOSμ knockout and wild type mice suggests that nNOSβ, which is present in both genotypes, could be responsible for NO-mediated skeletal muscle glucose uptake during contraction.