An ideal scaffold for cartilage tissue engineering should be biomimetic in not only its biochemical composition, but also in the morphological structure of the scaffold. In this study, we fabricated a scaffold with an oriented structure using a nanofibrous articular cartilage extracellular matrix (ACECM), in which the ACECM was used to mimic the biochemical composition and oriented structure of articular cartilage. Histology analysis showed that the scaffold contained cartilage ECM (GAGs and collagen II). Scanning electron microscopy (SEM) indicated that the scaffolds were composed of nanofibers and possessed vertical microtubules. Chondrogenic differentiation-induced mesenchymal stem cells (MSCs) were seeded on the scaffold in vitro. SEM showed that MSCs proliferated well and aligned along the vertical microtubules, which mimicked the orientation of deep zone articular cartilage. A cell proliferation assay and live/dead cell staining demonstrated that the ACECM possessed good cell affinity, which favored cell adherence and proliferation. The MSCs that had been labeled with the fluorescent dye PKH26 and seeded on scaffolds were implanted into nude mice. The differentiated cells/ACECM implants formed cartilage-like tissue 4 weeks after implantation, and stained positive for collagen type II and toluidine blue. In addition, the in vivo fluorescent images verified that the MSCs in the implants were the labeled MSCs. These results demonstrated that the oriented ACECM scaffolds hold great promise for use in cartilage tissue engineering applications.