Insulin resistance of skeletal muscle is a major defect in obesity and type 2 diabetes. Insulin resistance has been associated with a chronic sub-clinical inflammatory state in epidemiological studies and specifically with activation of the Inhibitor -B kinase (IBK)- Nuclear factor -B (NFB) pathway. However it is unclear whether this pathway plays a role in mediating insulin resistance in muscle in vivo. We separately over-expressed the p65 sub-unit of NFB and IBK in single muscles of rats using in vivo electrotransfer and compared the effects after one week versus paired contralateral control muscles. A 64% increase in p65 protein (p<0.001) was sufficient to cause muscle fibre atrophy but had no effect on glucose disposal or glycogen storage in muscle under hyperinsulinaemic-euglycaemic clamp conditions. Similarly a 650% increase in IBK expression (p<0.001) caused a significant reduction in Inhibitor -B protein but also had no effect on clamp glucose disposal after lipid infusion. In fact, IBK over-expression in particular caused increases in activating tyrosine phosphorylation of Insulin Receptor Substrate-1 (24%; p=0.02) and serine phosphorylation of Akt (23%; p<0.001), implying a moderate increase in flux through the insulin signalling cascade. Interestingly, p65 over-expression resulted in a negative feedback reduction of 36% in Toll-like Receptor (TLR)-2 (p=0.03) but not TLR-4 mRNA. In conclusion activation of the IBK-NFB pathway in muscle does not seem to be an important local mediator of insulin resistance.