The electrical conductivity of graphene provides a unique opportunity to modify the behavior of electrically sensitive cells. Here, we demonstrate that C2C12 myoblasts that were cultured on ultrathin thermally reduced graphene (TR-Graphene) films had more favorable cell adhesion and spreading compared to those on graphene oxide (GO) and glass slide substrates, comparable with conventional Petri dish. More importantly, we demonstrate that electrical stimulation significantly enhanced myoblast cell differentiation on a TR-Graphene substrate compared to GO and glass slide surfaces as confirmed by the expression of myogenic genes and proteins. These results highlight the potential applications of graphene-based materials for cell-based studies, bioelectronics, and biorobotics. © 2014 The Royal Society of Chemistry.