Objective Insulin resistance and hyperinsulinemia are the hallmark features of noninsulin-dependent diabetes mellitus (NIDDM) and obesity, but the detailed pathogenesis underlying the initiation of insulin resistance is still poorly understood. Recently, fibroblast growth factor 21 (FGF21), a novel member of the FGF family, was identified as a potent metabolic regulator with specific effects on glucose and lipid metabolism. We investigated the mechanisms involved in the induction of FGF21 resistance in human skeletal muscle of type 2 diabetes (T2DM) patients. Methods We recruited 16 type 2 diabetic subjects and 12 subjects with normal glucose tolerance for this study. All subjects signed informed consent forms regarding the experimental procedures. The Institutional Medical Ethics Committee approved all methods. Whole-body insulin-mediated glucose uptake was determined using a euglycemic hyperinsulinemic clamp test. A percutaneous biopsy sample of the vastus lateralis muscle was obtained from 15 to 20 cm above the knee using a Bergstrom needle. Differentiated HSMMs were pretreated with palmitate at three different concentrations for 24 h, and then were stimulated for 10 min with recombinant FGF21 (100 ng/mL). We measured levels of FGF21, FGF Receptor, FRS2, and ERK1/2 using antibodies specific for FGF21 (Abcam, Cambridge, MA), phospho-FGF Receptor (Tyr653/654), phospho r-FRS2-α (Tyr196), and phospho-ERK1/2 (Thr202/Tyr 204) (Cell Signaling technology, Danvers, MA), respectively. Results Levels of FGF21 were significantly increased in skeletal muscles from T2DM subjects but levels of active FGF receptor and FRS2α were significantly decreased in skeletal muscles from T2DM subjects. Similarly, FGF21 increased levels of active FGF receptor, FRS2α and ERK1/2 in HSMMs but there were significantly decreased levels of FGF21-stimulated FGF receptor, FRS2α and ERK1/2 in HSMMs treated with palmitate. Conclusion In conclusion, FGF21 resistance apparently was involved in insulin resistance in skeletal muscle in type 2 diabetes.