Many musculoskeletal tissues exhibit significant anisotropic mechanical properties reflective of a highly oriented underlying extracellular matrix. For tissue engineering, recreating this organization of the native tissue remains a challenge. To address this issue, this study explored the fabrication of biodegradable nanofibrous scaffolds composed of aligned fibers via electrospinning onto a rotating target, and characterized their mechanical anisotropy as a function of the production parameters. The characterization showed that nanofiber organization was dependent on the rotation speed of the target;​ randomly oriented fibers (33% fiber alignment) were produced on a stationary shaft, whereas highly oriented fibers (94% fiber alignment) were produced when rotation speed was increased to 9.3 m/s. Non-aligned scaffolds had an isotropic tensile modulus of 2.1±0.4 MPa, compared to highly anisotropic scaffolds whose modulus was 11.6±3.1 MPa in the presumed fiber direction, suggesting that fiber alignment has a profound effect on the mechanical properties of scaffolds. Mechanical anisotropy was most pronounced at higher rotation speeds, with a greater than 33-fold enhancement of the Young's modulus in the fiber direction compared to perpendicular to the fiber direction when the rotation speed reached 8 m/s. In cell culture, both the organization of actin filaments of human mesenchymal stem cells and the cellular alignment of meniscal fibroblasts were dictated by the prevailing nanofiber orientation. This study demonstrates that controllable and anisotropic mechanical properties of nanofibrous scaffolds can be achieved by dictating nanofiber organization through intelligent scaffold design.