In the present study, we reported the metabolic effects of salidroside, one of the active components of Rhodiola Rosea, on skeletal muscle cells. Salidroside dose-dependently stimulated glucose uptake in differentiated L6 rat myoblast cells. Inhibitor of AMP-activated protein kinase (AMPK) by pretreating the cells with compound C potently reduced salidroside-stimulated glucose uptake, while inhibition of phosphatidylinositol 3-kinase (PI3K) by wortmannin exhibited no significant inhibitory effect on salidroside-mediated glucose transport activation. Western blotting analyses revealed that salidroside increased the phosphorylation level of AMPK and acetyl-CoA carboxylase (ACC). In addition, salidroside enhanced insulin-mediated Akt activation and glucose uptake, and such enhancement can be specifically inhibited by compound C. In summary, AMPK activation was involved in the effects of salidroside on glucose transport activation and insulin sensitivity. Salidroside can be further developed as potential compound for the anti-diabetic therapy.