This study aims to elucidate the role of Transcription factor EB (TFEB) in protecting C2C12 myotubes against palmitate (PA)-induced insulin resistance (IR) and explored its mechanism associated with autophagy. PA treatment significantly decreased insulin sensitivity in myotubes and downregulated TFEB protein expression. TFEB overexpression significantly reversed the PA-suppressed glucose transporter 4 (GLUT4) protein expression and improved intracellular glucose uptake and consumption, and also alleviated the decrease of authphagy markers induced by PA. The effect of TFEB overexpression on GLUT4 was also abolished by the autophagy inhibitor 3-MA. In addition, AMPKɑ2-DN inhibited or abolished the effects of TFEB overexpression on upregulation of GLUT4 and PA-induced decrease of autophagy marker expressions. Taken together, our data demonstrated that upregulation of TFEB improved PA-induced IR in C2C12 myotubes by enhancing autophagy and upregulating AMPK activity. TFEB, as a critical regulator of glucose homeostasis in skeletal muscle cells, may be a potential therapeutic target for IR and Type 2 diabetes.