Toll-like receptors (TLRs) are transmembrane proteins that detect a variety of molecular components mostly derived from microorganisms. TLR2 and TLR4 are amongst others present in liver, adipose tissue, and skeletal muscle. Extracellular long-chain fatty acids bind TLR2 and 4 and induce downstream signalling cascades implicated in cellular stress and inflammatory processes. Evidence indicates that TLR activation by non-esterified fatty acids (NEFAs) may participate in the development of insulin resistance. Exercise seems to induce a downregulation of TLR expression in various tissues, a mechanism that may take part in the protective effect of exercise against insulin resistance. Moreover, TLRs seem to mediate the activation of mitogen-activated protein kinase p38 and Jun-amino-terminal kinase by extracellular NEFAs during endurance exercise. During this type of exercise, circulating NEFAs are known to regulate the expression of various genes including pyruvate dehydrogenase kinase 4, uncoupling protein 3, carnitine palmitoyltransferase 1, and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha. Whether these events are initiated by a TLR-dependent signal transduction remains to be investigated.