Reconstruction of the anisotropic structure and proper function of the knee meniscus remains an important challenge to overcome, because the complexity of the zonal tissue organization in the meniscus has important roles in load bearing and shock absorption. Current tissue engineering solutions for meniscus reconstruction have failed to achieve and maintain the proper function in vivo because they have generated homogeneous tissues, leading to long-term joint degeneration. To address this challenge, we applied biomechanical and biochemical stimuli to mesenchymal stem cells seeded into a biomimetic scaffold to induce spatial regulation of fibrochondrocyte differentiation, resulting in physiological anisotropy in the engineered meniscus. Using a customized dynamic tension-compression loading system in conjunction with two growth factors, we induced zonal, layer-specific expression of type I and type II collagens with similar structure and function to those present in the native meniscus tissue. Engineered meniscus demonstrated long-term chondroprotection of the knee joint in a rabbit model. This study simultaneously applied biomechanical, biochemical, and structural cues to achieve anisotropic reconstruction of the meniscus, demonstrating the utility of anisotropic engineered meniscus for long-term knee chondroprotection in vivo.