Introduction: Paralytic strabismus involves a functional loss of extraocular muscles resulting from muscular or neuronal disorders. Currently, only a limited number of drugs are available for functional repair of extraocular muscles. Here, we investigated the effects of a novel drug, flavonoids sophoranone, on the differentiation of extraocular muscles as assessed in bothin vivo and in vitro models. Materials and Methods: The effect of flavonoids sophoranone on C2C12 cells was examinedin vitro as evaluated with use of apoptosis, reactive oxygen species (ROS), and cell viability assays. Then, both in vivo and in vitro effects of this drug were examined on the differentiation of C2C12 and satellite cells within extraocular muscles in rabbits. For these latter experiments, RT-PCR and Western blot assays were used to determine expression levels of markers for myogenic differentiation. Results: With use of flavonoids sophoranone concentrations ranging from 0 to 10 μM, no effects were observed upon cell apoptosis, ROS, and cell cycle in C2C12 cells. Based on MTT assay results, flavonoids sophoranone was shown to increase C2C12 cell proliferation. Moreover, flavonoids sophoranone promoted the differentiation of C2C12 and satellite cells within extraocular muscles in rabbits, which were verified as based on cell morphology and expression levels of mRNA and protein markers of myogenic differentiation. Finally, flavonoids sophoranone treatment also increased gene expressions of Myh3, Myog, and MCK. Conclusion: The capacity for flavonoids sophoranone to upgrade the differentiation of both C2C12 and satellite cells within extraocular muscles in rabbits at concentrations producing no adverse effects suggest that this drug may provide a safe and effective means to promote repair of damaged extraocular muscles.