The aim of this study is to investigate the effects of scaffold structure on ligament tissue engineering. In previous ligament tissue engineering studies, the advantages of aligned electrospun fibrous scaffold and cyclic loading have been demonstrated. In the current study, we constructed structures by sandwiching fibroblasts with two electrospun microfiber scaffolds and applied cyclic stretching using a custom-design mechanical stretching bioreactor. Through the dynamic tensile stimulation, different results were observed on the fibroblasts seeded on different structures. In addition, the effects of tensile strain and shear strain on the ACL fibroblasts were also examined. Cells were subjected to different scaffold structures (parallel, opposing, and random), normal strain and shear strain, which were designed to mimic the native environment experienced by ligament fibroblasts in vivo. After the two-week culturing period, DNA, collagen, and GAGs contents and mechanical property were analyzed. Cell morphology was affected by the scaffold structures. Cyclic mechanical stimulation did not affect cell proliferation after two weeks of culture. However, collagen synthesis increased with dynamic loading in the opposing fiber structure group. The increase of collagen interestingly did not correspond with an increase in DNA content, indicating that the collagen synthesis per cell was increased, suggesting the involvement of stretch-induced shear strain on the opposing fibers. After 4 weeks of culturing, the cell proliferation was not enhanced by dynamic loading, it did showed effectiveness in increasing collagen synthesis . This result suggested that the principal strain also benefit on ligament fibroblast, though the benefit of stretch-induced shear strain on ligament fibroblast seems occurred earlier.