Native heart valve leaflets with layered fibrous structures show anisotropic characteristics, allowing them to withstand complex mechanical loading for long-term cardiac cycles. Herein, two types of silk fibroin (SF) fiber membranes with anisotropic (ASF) and isotropic (ISF) properties were prepared by electrospinning, and were further combined with poly(ethylene glycol) diacrylate (PEGDA) hydrogels to serve as polymeric heart valve (PHV) substitutes (PEGDA-ASF and PEGDA-ISF). The uniaxial tensile tests showed obvious anisotropy of PEGDA-ASF with elastic moduli of 10.95±1.09 and 3.55±0.32 MPa, respectively, along the directions parallel and perpendicular to the fiber alignment, while PEGDA-ISF possessed isotropic property with elastic moduli of 4.54± 0.43 MPa. The PHVs from both PEGDA-ASF and PEGDA-ISF presented appropriate hydrodynamic properties from pulse duplicator tests according to the ISO 5840-3 standard. However, finite element analysis (FEA) revealed the anisotropic PEGDA-ASF valve showed a lower maximum principle stress value (2.20 MPa) in commissures during diastole compared with that from the isotropic PEGDA-ISF valve (2.37 MPa). In the fully open state, the bending area of the PEGDA-ASF valve appeared in the belly portion and near the attachment line like native valves, however, which was close to free edges for the PEGDA-ISF valve. The Gauss curvature analysis also indicated that the anisotropic PEGDA-ASF valve can produce appropriate surface morphology by dynamically adjusting the movement of bending area during the opening process. Hence, anisotropy of PHVs with bio-inspired layered fibrous structures played important roles in mechanical and hydrodynamic behavior mimicking native heart valves.