Rho guanine nucleotide exchange factor 9 (ARHGEF9), as a protein that assists small GTPases, is widely present in various tissues. It has been reported to play an important role mainly in neurological diseases and gliomas. However, there have been no reports on its impact on skeletal muscle regeneration after injury. This study first demonstrated a significant increase in ARHGEF9 protein expression during the regeneration of skeletal muscle post-injury in mice. Secondly, during the differentiation of mouse C2C12 myoblasts, ARHGEF9 significantly increased and co-localized with actin filaments. Inhibition of ARHGEF9 significantly downregulated the migration rate and actin filaments polymerization of mouse C2C12 myoblasts, and significantly reduced the expression of proteins related to cell migration. Finally, inhibition of ARHGEF9 significantly reduced the differentiation ability of mouse C2C12 myoblasts. In summary, ARHGEF9 impacting on myoblasts migration and differentiation suggests that targeting ARHGEF9 could be beneficial for promoting skeletal muscle regeneration and repair.