BACKGROUND AND PURPOSE: Endoplasmic reticulum (ER) stress has been implicated in the pathogeneses of insulin resistance and type 2 diabetes, and extracellular signal-regulated kinase (ERK) antagonist is an insulin sensitizer that can restore muscle insulin responsiveness in both tunicamycin-treated muscle cells and type 2 diabetic mice. The present study was undertaken to determine whether the chemical or genetic inhibition ER stress pathway targeting by ERK results in metabolic benefits in muscle cells. EXPERIMENTAL APPROACH: ER stress was induced in L6 myotubes using tunicamycin (5 μg·mL(-1) ) or thapsigargin (300 nM) and cells were transfected with siRNA ERK or AMPKα2. Changes in ER stress and in the ERK and AMPK signalling pathways were explored by Western blotting. The phosphorylation levels of insulin receptor substrate 1 were analysed by immunoprecipitation and using glucose uptake assay. KEY RESULTS: ER stress dampened insulin-stimulated signals and glucose uptake, whereas treatment with the specific ERK inhibitor U0126 (25 μM) rescued impaired insulin signalling via AMPK activation. In db/db mice, U0126 administration decreased markers of insulin resistance and increased the phosphorylations of Akt and AMPK in muscle tissues. CONCLUSIONS AND IMPLICATIONS: Inhibition of ERK signalling pathways by a chemical inhibitor and knockdown of ERK improved AMPK and Akt signallings and reversed ER stress-induced insulin resistance in L6 myotubes. These findings suggest that ERK signalling plays an important role in the regulation of insulin signals in muscle cells under ER stress. Abbreviations: ACC, acetyl-CoA carboxylase; Ad-DN-AMPK adenovirus dominat negative; AMPK, AMP-activated protein kinase; ER, endoplasmic reticulum; PERK, RNA-activated protein kinase-like ER resident kinase; IRE-1, inositol-requiring kinase-;siRNA, small interfering RNA; UPR, unfolded protein; IRS-1, insulin receptor substrate-1; WT, wild type