Objective：Aiming at the problem of significant anisotropy in the 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 with 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 anisotropy of material extrusion has a significant effect on the mechanical properties of the 3D printed bone substitute, and the long axis is recommended to be perpendicular to the building platform during the 3D printing of the substitute of the load-bearing long bone.