Elevated free fatty acid (FFA) is a key risk factor for insulin resistance (IR). Our previous studies found that mangiferin could decrease serum FFA levels in obese rats induced by a high-fat diet. Our research was to determine the effects and mechanism of mangiferin on improving IR by regulating FFA metabolism in HepG2 and C2C12 cells. The model was used to quantify PA-induced lipid accumulation in the two cell lines treated with various concentrations of mangiferin simultaneously for 24 h. We found that mangiferin significantly increased insulin-stimulated glucose uptake, via phosphorylation of protein kinase B (P-AKT), glucose transporter 2 (GLUT2), and glucose transporter 4 (GLUT4) protein expressions, and markedly decreased glucose content, respectively, in HepG2 and C2C12 cells induced by PA. Mangiferin significantly increased FFA uptake and decreased intracellular FFA and triglyceride (TG) accumulations. The activity of the peroxisome proliferator-activated receptor α (PPARα) protein and its downstream proteins involved in fatty acid translocase (CD36) and carnitine palmitoyltransferase 1 (CPT1) and the fatty acid β-oxidation rate corresponding to FFA metabolism were also markedly increased by mangiferin in HepG2 and C2C12 cells. Furthermore, the effects were reversed by siRNA-mediated knockdown of PPARα. Mangiferin ameliorated IR by increasing the consumption of glucose and promoting the FFA oxidation via the PPARα pathway in HepG2 and C2C12 cells.