Toll-like receptor 2 (TLR2) and 4 (TLR4) are present in the plasma membrane of skeletal muscle where their functions are only partially understood. A variety of molecular components could activate TLR2 and TLR4, including non-esterified fatty acids (NEFA). NEFA, also are known to be released during endurance exercise. Likewise, endurance exercise may activate know downstream targets of TLR2 and TLR4 such as mitogen- activated protein kinase (MAPK) and nuclear factor-ĸB (NF-ĸB). The purpose of the present work was to investigate the plausible interaction between elevated plasma NEFA observed during endurance exercise and the MAPK activation mediated by TLR2 and TLR4. From a methodological point of view, three experimental models were used: myotubes cultured from C2C12 cells, soleus and tibialis muscle from C57BL/6J animals wild-type (WT) and transgenic tlr2-/- and tlr4-/- mice, and finally, muscle biopsies taken from the vastus lateralis of healthy and diabetics subjects. Our data supports a role for plasma NEFA in the activation of MAPK during endurance exercise. This activation may be mediated by TLR2 and TLR4 in skeletal muscle.