Reduced in insulin-induced glucose uptake in skeletal muscles is a hallmark of insulin resistance. This study investigated the effects of Mesembryanthemum crystallinum extract (MCE) on palmitate-induced insulin resistance in C2C12 skeletal muscle cells. MCE reversed the palmitate-induced inhibition of glucose uptake, suppressed the phosphorylation of insulin receptor substrate-1 (IRS-1), and significantly promoted the activation of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), glucose transporter-4 (GLUT4) and AMP-activated protein kinase (AMPK). Furthermore, MCE treatment of C2C12 cells inhibited palmitate-induced intracellular lipid accumulation and increased the expression of peroxisome proliferator-activated receptor (PPAR-γ). These findings suggest that MCE may improve palmitate-induced insulin resistance in skeletal muscle cells, indicating that MCE treatment could be beneficial for patients with diabetes.