Lauric acid (LA), which is the primary fatty acid in coconut oil, was reported to have many metabolic benefits. TLR4 is a common receptor of lipopolysaccharides and involved mainly in inflammation responses. Here, we focused on the effects of LA on skeletal muscle fiber types and metabolism. We found that 200 mu M LA treatment in C2C12 or dietary supplementation of 1% LA increased MHCIIb protein expression and the proportion of type lib muscle fibers from 0.452 +/- 0.0165 to 0.572 +/- 0.0153, increasing the mRNA expression of genes involved in glycolysis, such as HK2 and LDH2 (from 1.00 +/- 0.110 to 1.35 +/- 0.0843 and from 1.00 +/- 0.123 to 1.71 +/- 0.302 in vivo, respectively), decreasing the catalytic activity of lactate dehydrogenase (LDH), and transforming lactic acid to pyruvic acid. Furthermore, LA activated TLR4 signaling, and TLR4 knockdown reversed the effect of LA on muscle fiber type and glycolysis. Thus, we inferred that LA promoted glycolytic fiber formation through TLR4 signaling.