It has been demonstrated that mechanical stimulation plays a vital role in regulating the proliferation and differentiation of stem cells. However, little is known about the effects of mechanical stress on tendon/ligament development from mesenchymal stem cells (MSCs). Here, using a custom-made cell-stretching device, we studied the effects of mechanical stretching on the cell morphology and mRNA expression of several key genes modulating tendon/ligament genesis. We demonstrate that bone-marrow-derived rat MSCs (rMSCs), when subjected to cyclic uniaxial stretching, express obvious detectable mRNAs for tenascin C and scleraxis, a unique maker of tendon/ligament formation, and significantly increased levels of type I collagen and type III collagen mRNAs. The stretched cells also orient at approximately 65 degrees with respect to the stretching direction and exhibit a more fibroblast-like morphology. Collectively, these results indicate that mechanical stretching facilitates the directed differentiation of rMSCs into tendon/ligament fibroblasts, which has potential implications for the tissue engineering of bioartificial tendons and ligaments.