Osteoarthritis affects approximately 500 million individuals globally, with severe cases often leading to osteochondral defects. Biomimetic collagen-hydroxyapatite scaffolds have been investigated for the treatment of osteochondral defects. However, achieving precise mimicry of the intricate composition, gradient nanostructure, and biological function of native tissue remains a formidable challenge. Drawing inspiration from the native osteochondral tissue, we present BioGradCart, an innovative mineralized collagen scaffold featuring multiple gradients designed for osteochondral regeneration. Comprising gradient collagen fibers and mineralized hydroxyapatite, this scaffold demonstrates a mechanical gradient and maintains a stable interface from hyaline cartilage to subchondral bone. Notably, the BioGradCart scaffold possesses enhanced biocompatibility and bioactivity, significantly facilitating gradient differentiation of bone marrow-derived stem cells towards chondrogenic and osteogenic phenotypes. In a male rabbit model with osteochondral defects, the scaffold manifests a remarkable capacity to accelerate osteochondral regeneration, promote the formation and alignment of collagen fibers. However, further investigation is required to evaluate its efficacy in a female animal model. The development of the BioGradCart scaffold provides a promising platform for advancing the management of osteochondral defects and osteoarthritis.