Muscle contractile activity functions as a potent stimulus for acute interleukin (IL)-6 expression in working skeletal muscles. Recently, we established an "in vitro contraction model" using highly-developed contractile C2C12 myotubes by applying electric pulse stimulation (EPS). Herein, we characterize the effects of EPS-evoked contraction on IL-6 expression in contractile C2C12 myotubes. Both secretion and mRNA expression of IL-6 were significantly upregulated by EPS in a frequency-dependent manner in contracting myotubes during a 24-h period, and the response was blunted by cyclosporine A, a calcineurin inhibitor. Longer time (~12h) was required for the induction of IL-6 after the initiation of EPS as compared to that of other contraction-inducible CXC chemokines such as CXCL1/KC, which were induced in less than 3 hours. Furthermore, these acute inducible CXC chemokines exhibited no autocrine effect on IL-6 expression. Importantly, contraction-dependent IL-6 up-regulation was markedly suppressed in the presence of high levels of glucose along with increased glycogen accumulations. Experimental manipulation of intracellular glycogen contents by modulating available glucose or pyruvate during a certain EPS period further established the suppressive effect of glycogen accumulations on contraction-induced IL-6 up-regulation, which appeared to be independent of calcineurin activity. We also document that EPS-evoked contractile activity improved insulin-responsiveness in terms of intracellular glycogen accumulations. Taken together, these data provide important insights into the regulation of IL-6 expression in response to contractile activity of muscle cells, which is difficult to examine using in vivo experimental techniques. Our present results thus expand the usefulness of our "in vitro contraction model".