Leukemia inhibitory factor (LIF) is a multifunctional cytokine belonging to the interleukin-6 family and has been shown to stimulate regeneration of injured skeletal muscle. Although LIF has been shown to stimulate muscle cell proliferation, its precise role in differentiation is unclear. Thus, we examined the effect of LIF on the differentiation of cultured C2C12 myoblast cells. In this study, we used both non-glycosylated LIF expressed in bacteria and glycosylated LIF secreted from NIH3T3 cells infected with Ad-LIF. Both non-glycosylated and glycosylated LIF blocked differentiation of myoblasts as measured by expression of myosin heavy chain and myotube formation. Treatment of myoblasts with LIF induced phosphorylation of ERK, and the LIF-induced inhibitory effect on myogenesis was blocked by pretreatment with U0126, a specific MEK inhibitor, and transient transfection with dominant negative (DN)-MEK1. In contrast, although LIF activated STAT3, the LIF-induced repression of the MCK transcriptional activity was not reversed by pretreatment with AG490, a specific Jak kinase inhibitor or transient transfection with DN-STAT3. Additionally, LIF exhibited its inhibitory effect on myogenesis only when cells were treated at earlier than 12 h after inducing differentiation. Taken together, these results suggest that LIF strongly inhibited early myogenic differentiation though activation of the ERK signaling pathway and its effect is irrespective of glycosylation.