Endoplasmic reticulum stress has been linked to insulin resistance in multiple tissues but the role of endoplasmic reticulum stress in skeletal muscle has not been explored. Endoplasmic reticulum stress has been reported to increase tribbles 3 expression in multiple cell lines. Here, we report that high-fat feeding in mice, and obesity and type 2 diabetes in humans significantly increases tribbles 3 and endoplasmic reticulum stress markers in skeletal muscle. Overexpression of tribbles 3 in C2C12 myotubes and mouse tibialis anterior muscles significantly impairs insulin signalling. Incubation of C2C12 cells and mouse skeletal muscle with endoplasmic reticulum stressors thapsigargin and tunicamycin increases tribbles 3 and impairs insulin signalling and glucose uptake, effects reversed in cells overexpressing RNAi for tribbles 3 and in muscles from tribbles 3 knockout mice. Furthermore, tribbles 3 knockout mice are protected from high-fat diet-induced insulin resistance in skeletal muscle. These data demonstrate that tribbles 3 mediates endoplasmic reticulum stress-induced insulin resistance in skeletal muscle.