Toll-like receptor 4 (TLR4), a protein integral to innate immunity, is elevated in skeletal muscle of obese and type 2 diabetic humans and has been implicated in the development of lipid-induced insulin resistance. The purpose of this study was to examine the role of TLR4 as a modulator of basal (non-insulin-stimulated) substrate metabolism in skeletal muscle with the hypothesis that its activation would result in reduced fatty acid oxidation and increased partitioning of fatty acids toward neutral lipid storage. Human skeletal muscle, rodent skeletal muscle, and skeletal muscle cell cultures were employed to study the functional consequences of TLR4 activation on glucose and fatty acid metabolism. Herein, we demonstrate that activation of TLR4 with low (metabolic endotoxemia) and high (septic conditions) doses of LPS results in increased glucose utilization and reduced fatty acid oxidation in skeletal muscle and that these changes in metabolism in vivo occur in concert with increased circulating triglycerides. Moreover, animals with a loss of TLR4 function possess increased oxidative capacity in skeletal muscle and present with lower fasting levels of triglycerides and nonesterified free fatty acids. Evidence is also presented to suggest that these changes in substrate metabolism under metabolic endotoxemic conditions are independent of skeletal muscle-derived proinflammatory cytokine production. This report illustrates that skeletal muscle is a target for circulating endotoxin and may provide critical insight into the link between a proinflammatory state and dysregulated metabolism as observed with obesity, type 2 diabetes, and metabolic syndrome.