Obesity is associated with insulin resistance and a state of abnormal inflammatory response. The Toll-like receptor (TLR)4 has an important role in inflammation and immunity, and its expression has been reported in most tissues of the body, including the insulin-sensitive ones. Because it is activated by lipopolysaccharide and saturated fatty acids, which are inducers of insulin resistance, TLR4 may be a candidate for participation in the cross-talk between inflammatory and metabolic signals. Here, we show that C3H/HeJ mice, which have a loss-of-function mutation in TLR4, are protected against the development of diet-induced obesity. In addition, these mice demonstrate decreased adiposity, increased oxygen consumption, a decreased respiratory exchange ratio, improved insulin sensitivity, and enhanced insulin-signaling capacity in adipose tissue, muscle, and liver compared with control mice during high-fat feeding. Moreover, in these tissues, control mice fed a high-fat diet show an increase in IκB kinase complex and c-Jun NH₂-terminal kinase activity, which is prevented in C3H/HeJ mice. In isolated muscles from C3H/HeJ mice, protection from saturated fatty acid–induced insulin resistance is observed. Thus, TLR4 appears to be an important mediator of obesity and insulin resistance and a potential target for the therapy of these highly prevalent medical conditions.