Mechano-growth factor (MGF), the insulin growth factor- I (IGF-I) splice variant, has drawn an increasing attention in recent years. In this study, using a newly established system, we three-dimensionally (3D) cultured rat skeletal myoblasts and loaded them with cyclic uniaxial tensile strain of different magnitudes or time duration. After that, the cell proliferative index (PI) and mRNA expression of MGF, IGF-IEa and integrin beta1 were assayed. The major findings are: (1) mechanical stimulation induced MGF upregulation commensurate with cell PI elevation both in the 3D and 2D cultured myoblasts, but stronger mechanical force was needed to activate MGF expression in the 3D cultures; (2) MGF but not IGF-IEa was essential for mechano-induced myoblast replication, as IGF-IEa upregulation lagged cell PI elevation; and (3) the time course upregulation of MGF and integrin beta1 was correlated with each other, suggesting they shared a common mechanotransduction pathway.