In this work, three layers of human aorta, i.e. intima, media and adventitia, obtained from a 9 mm CryoValve® aortic valve allograft, were tested along both axial and circumferential directions. Preconditioning with a stretch ratio of 1.35 was used to mimic the physiological pulsatile loading conditions of the tissue. Results suggested that the stiffness along circumferential direction is generally larger than axial direction in each of the three layers. In all three layers, the media layer is the stiffest, and the adventitia layer is the softest regardless of testing directions. The anisotropic, nonlinear elastic mechanical behavior of each layer from the aortic valve allograft could help better understand the compliance mismatch between the allograft and native arterial tissue.