Extracellular matrix (ECM) materials have diverse physiological functions by themselves and can also act as reservoirs of cytokines and growth factors, so that they can affect the cell phenotype, attachment, migration and proliferation of cells. In this study, an ECM scaffold derived from porcine cartilage was evaluated for whether it can support and maintain chondrogenesis of rabbit mesenchymal stem cells (rMSCs) in vitro and in the nude mouse model in vivo. The porcine ECM scaffold was compared to a polyglycolic acid (PGA) scaffold and an MSC pellet as a control group. In an in vitro environment until 4 weeks, the ECM scaffold evoked chondrogenic differentiation of rMSCs earlier and produced more cartilaginous tissues than the PGA scaffold. Next, rMSCs in each scaffold were preconditioned with chondrogenic media in vitro for 1 week and implanted in the backs of nude mice for 6 weeks. The initially formed cartilaginous tissues turned into bone matrix with time centripetally from the outside of the region as observed by Safranin-O and von Kossa stains. This phenomenon progressed much more rapidly in the PGA group than in the ECM group. In the ECM group, the chondrogenic phenotypes of rMSCs were also maintained longer than in the PGA group. The loss of chondrogenic phenotypes was accompanied by the calcification of matrix, and hypertrophic changes by immunohistochemistry for osteocalcin and collagen type I and X. Blood vessel invasion took place more deeply and intensively in the PGA group. These results suggest that the ECM scaffold not only strongly supports chondrogenic differentiation of rMSCs, but also helps maintain its phenotype in vivo. We speculate that the ECM scaffold provides rMSCs with a favorable, native cartilage-like environment and therefore can be a promising tool for cartilage tissue engineering.