Endoplasmic reticulum (ER) stress is a central actor in the physiopathology of insulin resistance (IR) in various tissues. The subsequent unfolded protein response (UPR) interacts with insulin signaling through inositol‐requiring 1α (IRE1α) activation and tribbles homolog 3 (TRB3) expressions. IRE1α impairs insulin actions through the activation of c‐Jun N‐terminal kinase (JNK), and TRB3 is a pseudokinase inhibiting Akt. In muscle cells, the link between ER stress and IR has only been demonstrated by using chemical ER stress inducers or overexpression techniques. However, the involvement of ER stress in lipid‐induced muscle IR remains controversial. The aim of the study is to test whether palmitate‐induced IRE1α signaling and TRB3 expression disturb insulin signaling in myogenic cells. C2C12 myotubes were exposed to palmitate and then stimulated with insulin. siRNA transfection was used to downregulate TRB3 and IRE1α. Palmitate increased TRB3 expression, activated IRE1α signaling, and reduced the insulin‐dependent Akt phosphorylation. Knocking down TRB3 or IRE1α did not prevent the inhibitory effect of palmitate on Akt phosphorylation. Our results support the idea that ER stress is not responsible for lipid‐induced IR in C2C12 myotubes.