Histone lysine demethylation, as one of the most important mediator in the regulation of transcription, is associated with a various physiological status. KDM2B is a novel histone H3K4 and H3K36 demethylase involved in transcriptional activation. Here, we provide evidence for a unique dimension to epigenetic regulation of skeletal myogenesis. KDM2B is expressed abundantly in C2C12 myoblast cells and the expression levels of mRNA and protein gradually decreases during myogenic differentiation. Forced expression of KDM2B inhibits skeletal muscle differentiation. Conversely, knockdown of KDM2B by siRNA significantly induces muscle cell differentiation. KDM2B interacts with SRF to regulate expression of MRFs in C2C12 myoblast cells. SRF is demethylates by KDM2B and directly demethylates it at lysine 165(K165) to constrain its transcriptional activity. Mutation of K165 prevents SRF-induced muscle differentiation. Together, these data indicate that KDM2B is a H3K4/K36 demethylase and plays an important role in the transcriptional regulation of muscle cell differentiation.