Mesenchymal stem cells (MSCs) are promising clinical tools, but the molecular mechanisms that regulate the mobilization and homing of MSCs and cause invasion through extracellular matrix (ECM) barriers are unknown. Matrix metalloproteinase (MMP) degrades the ECM and promotes cell migration. In this study, we investigated MMP expression and cell migration after treatment with erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF). Specifically, we studied whether EPO combined with G-CSF enhanced MMP expression and increased the in vitro motility of MSCs. Real-time PCR was used to detect the mRNA of MMPs and tissue inhibitors of metalloproteinase. Cell migration was evaluated by transwell and wound healing assays. Western blotting was used to detect changes in ERK1/2 protein levels. The results showed that EPO combined with G-CSF enhanced MMP-2 expression in MSCs, promoted MSC motility and activated the ERK1/2 signaling pathway. Thus, a combination treatment of EPO with G-CSF promoted cell migration by stimulating MMP-2 expression in MSCs and this appeared to be related to the ERK1/2 signaling pathway.