We examined optimal cyclic uniaxial stretches for stem cell-to-tenocyte differentiation by applying a wide range of cyclic mechanical stimuli. Human bone marrow mesenchymal stem cells (hBMSCs) were subjected to three types of cyclic elongation of 5%, 10%, or 15% at a cyclic frequency of 1 Hz for 24 h or 48 h, and differentiation into tenocytes was assessed by two methods: real-time polymerase chain reaction determination of gene expression levels and western blotting analysis of protein expression levels. The gene expression levels of the differentiation markers type I collagen (Col I), type III collagen (Col III), tenascin-C (Tnc), and scleraxis (Scx), all of which are constituents of tendon tissue, were increased when cells were exposed to 10% stretching stimulation. The levels of Col I and Tnc protein synthesis levels were also higher in the cells with 10% stretching stimulation than in those subjected to other stimuli. The results indicated that 10% stretching stimulus was efficient to induce the differentiation of hBMSCs into tenocytes. In addition, the changes in gene and protein expression levels were strongly correlated with cell orientation angle. The results presented here suggest that mesenchymal stem cell-to-tenocyte differentiation is strongly associated with cumulative elongation load on the cells. This work provides novel insights into the differentiation of tenogenesis in a strain-induced environment and supports the therapeutic potential of hBMSCs.