The purpose of this study was to explore the influences of cyclic mechanical stretching on the mRNA expressions of tendon/ligament-related and osteoblast-specific marker genes in human MSCs seeded onto a collagen type I-coated surface. The stretch-induced mRNA expressions of mesenchymal stem cell protein (MSCP), matrix metalloproteinase-3 (MMP-3), and marker genes related to tendon/ligament cells (type I collagen, type III collagen, and tenascin-C) and those typical of osteoblasts (core binding factor alpha 1 (Cbfa1), alkaline phosphatase (ALP), and osteocalcin (OCN)) were analyzed by quantitative real-time PCR. The results revealed significant downregulation of MSCP and upregulation of MMP-3 genes in MSCs subjected to mechanical loading, regardless of the magnitude of the stretching (high or low). Moreover, the typical marker genes of the osteoblast lineage were upregulated by low-magnitude stretching, whereas tendon/ligament-related genes were upregulated by high-magnitude stretching for a long period. Cbfa1 and ALP were upregulated starting as early at 8 hr, followed by a downward trend and no significant change in expression at the other time points. The mRNA expressions of type I collagen, type III collagen, and tenascin-C significantly increased in MSCs subjected to 10% stretching for 48 hr, and this effect still existed after the stretched cells had rested for 48 hr. This study demonstrated the effect of cyclic mechanical stretching on differential transcription of marker genes related to different cell lineages. Low-magnitude stretching increased mRNA expressions of Cbfa1 and ALP and was possibly involved in the early osteoblastic differentiation of MSCs, whereas high-magnitude stretching upregulated the mRNA expressions of tendon/ligament-related genes.