BACKGROUND AND OBJECTIVES: Bone marrow derived mesenchymal stem cells (BMSCs) have shown to be an appealing source for cell therapy and tissue engineering. Previous studies have confirmed that the application of low-level laser irradiation (LLLI) could affect the cellular process. However, little is known about the effects of LLLI on BMSCs. The aim of this study was designed to investigate the influence of LLLI at different energy densities on BMSCs proliferation, secretion and myogenic differentiation. STUDY DESIGN/MATERIALS AND METHODS: BMSCs were harvested from rat fresh bone marrow and exposed to a 635 nm diode laser (60 mW; 0, 0.5, 1.0, 2.0, or 5.0 J/cm(2)). The lactate dehydrogenase (LDH) release was used to assess the cytotoxicity of LLLI at different energy densities. Cell proliferation was evaluated by using 3-(4, 5-dimethylithiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assay. Production of vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) were measured by enzyme-linked immunosorbent assay (ELISA). Myogenic differentiation, induced by 5-azacytidine (5-aza), was assessed by using immunocytochemical staining for the expression of sarcomeric alpha-actin and desmin. RESULTS: Cytotoxicity assay showed no significant difference between the non-irradiated group and irradiated groups. LLLI significantly stimulated BMSCs proliferation and 0.5 J/cm(2) was found to be an optimal energy density. VEGF and NGF were identified and LLLI at 5.0 J/cm(2) significantly stimulated the secretion. After 5-aza induction, myogenic differentiation was observed in all groups and LLLI at 5.0 J/cm(2) dramatically facilitated the differentiation. CONCLUSIONS: LLLI stimulates proliferation, increases growth factors secretion and facilitates myogenic differentiation of BMSCs. Therefore, LLLI may provide a novel approach for the preconditioning of BMSCs in vitro prior to transplantation.