Objective Aiming at the problem of significant anisotropy in the three-dimensional ( 3D) printed polyether-ether-ketone ( PEEK) bone substitutes manufactured by material extrusion technology, taking the femur, the main load-bearing long bone of the lower limb, as an example, the biomechanical properties of the femoral model under different direction in the build chamber were evaluated by the combination of finite element analysis and in-vitro mechanical experiment. Methods A left femoral model was obtained by reconstruction from CT data. The stress and displacement of the 3D printed PEEK femur with different directions in the build chamber under five physiological postures in the human gait cycle were simulated by varying the orthogonal anisotropy mechanical properties. An in-vitro mechanical experiment was conducted to investigate the safety and stability of the femur through a 3D printed PEEK femur. Results When the long axis of the femur model was perpendicular to the building platform of the 3D printer, a better mechanical property was obtained, and the maximum von Mises stress was 46. 56 MPa, which was lower than the yield stress of PEEK, while the maximum displacement was larger than that of the natural femur under same loading condition. Therefore, the 3D printed PEEK femur met the strength requirement, but the stability needs to be improved. Conclusions The long axis is recommended to be perpendicular to the building platform when the material extrusion technology was used for the substitute of the load-bearing long bone, and the effect of its anisotropy on service performance of the substitute should be carefully considered when the 3D printing technology is used for load-bearing bone substitute.