Growing evidence suggests that obesity and associated metabolic dysregulation occurs in concert with chronic low-grade inflammation. Toll-like receptors (TLR) are transmembrane receptors that play an important role in innate immunity and the induction of inflammatory responses. Our laboratory has observed that TLR4 expression is elevated in the skeletal muscle of obese humans and is associated with reduced fatty acid (FA) oxidation and increased lipid synthesis. Additionally, activation of this pathway by lipopolysaccharide (LPS), ex vivo, results in a shift in substrate metabolism favoring glucose as an energy substrate and preferential storage of FA in intracellular lipid depots. The purpose of this study was to examine the effects of saturated vs. monounsaturated FA on TLR4 transcription and signaling using ex vivo and in vivo models. C2C12 myotubes were incubated in FA-enriched growth medium with varying ratios of palmitate and oleate for 12 hours. Following FA treatment, cells were either collected for measures of mRNA and protein levels of TLR4 or challenged with LPS (500 ng/mL) for 2 hours to assess TLR4 mediated changes in interleukin-6 (IL-6) and glucose and fatty acid metabolism. TLR4 mRNA and protein content were increased in stepwise fashion with higher palmitate concentration (p<0.05). This was associated with an exacerbated LPS effect on IL-6 mRNA and protein levels, and glucose and fatty acid metabolism. To determine if these effects are translated to an in-vivo model, C57BL/6 mice were fed high saturated fat (HSF), high monounsaturated fat (HMF), and control diets for 10 weeks. Following the dietary intervention, animals were challenged with I.P. injections of either saline or LPS (~25μg/mouse), sacrificed 4 hours post-injection, and red and white gastrocnemius muscle were harvested for measures of expression and protein levels of TLR4 and IL-6, and glucose and fatty acid metabolism. TLR4 mRNA and protein levels were not altered with either the HSF or HMF diets. However, there was a heightened LPS response with regards to increases in IL-6 and TNF-α, and enhanced shifts in substrate metabolism following the HSF diet (p<0.05). These effects were not observed in response to the HMF diet. In conclusion, these data demonstrate that a milieu of high saturated fatty acids results in elevated sensitization of the TLR4 pathway in skeletal muscle. These results provide insight into how a westernized diet, one enriched in saturated fat, may link chronic inflammation with obesity-associated metabolic abnormalities.