Mesenchymal stem cells (MSCs) have received considerable attention in regenerative medicine during the past decade. Eugenol is a natural and versatile vegetable molecule, which has a wide variety of therapeutic effects. Although different biological and pharmaceutical functions of Eugenol are well known, its effect on MSCs has not been studied yet. Therefore, this study was focused on investigating the effect of Eugenol on the proliferation and migration of bone marrow (BM)-derived MSCs in vitro. To do so, BM-MSCs were isolated from 4 to 8 weeks old NMRI mice. Cytotoxicity of Eugenol on MSCs was evaluated by MTT assay at 24, 48 and 72 h after treatment. In addition, its effect was assessed on the proliferation and migration of MSCs using wound healing assay in vitro and quantitative gene expression analysis for Oct4, Sox2, Cyclin-D1, Rex1, Tex10, Cxcr4, Vla4 and c-Met. Results showed that Eugenol reduced the number of MSCs in a dose- and time-dependent manner. The median inhibition concentration of Eugenol on MSCs was 400 μg/ml at 24 and 48 h and 200 μg/ml at 72 h after treatment. Moreover, about 90% viability of MSCs was detected at concentrations ≤12.5 μg/ml. The wound healing assay and gene expression analysis demonstrated that Eugenol promoted the migratory potential of MSCs through up-regulation of c-Met. Moreover, Eugenol has enhanced the proliferation of MSCs via over-expression of Sox2, Rex1 and Tex10. In conclusion, this study revealed that Eugenol enhances the proliferation and migration of MSCs, and thus this will be beneficial to the field of regenerative medicine.