Osteochondral defects pose an enormous challenge due to the lack of an effective repair strategy. To tackle this issue, the importance of a calcified cartilage interlayer (CCL) in modulating osteochondral regeneration should be valued. Herein, we proposed that an extracellular matrix (ECM) derived from a suitable cell source might efficiently promote the formation of calcified cartilage. To the end, cell sheets from four kinds of cells, including bone marrow mesenchymal stem cells (BMSCs), pre-osteoblasts (MC3T3), chondrocytes (Cho), and artificially induced hypertrophic chondrocytes (HCho), were obtained by seeding the cells on electrospun fibrous meshes, followed by decellularization to prepare decellularized ECMs (D-ECMs) for BMSC re-seeding and differentiation studies. For cell proliferation, the BMSC-derived D-ECM exhibited the strongest promotion effect. For inducing the hypertrophic phenotype of re-seeded BMSCs, both the BMSC-derived and HCho-derived D-ECMs demonstrated stronger capacity in up-regulating the depositions of related proteins and the expressions of marker genes, as compared to the MC3T3-derived and Cho-derived D-ECMs. Accordingly, from the histological results of their subcutaneous implantation in rats, both the BMSC-derived and HCho-derived D-ECMs displayed obvious Masson's trichrome and Safranin-O/Fast-Green staining colors simultaneously, representing the characteristics related to osteogenesis and chondrogenesis. Differently, MC3T3-derived and Cho-derived D-ECMs were mainly detected during the osteogenic or chondrogenic expression, respectively. These findings confirmed that the BMSC-derived D-ECM could induce hypertrophic chondrocytes, though being a little inferior to the HCho-derived D-ECM. Overall, the BMSC-derived D-ECM could be a potential material in constructing the interlayer for osteochondral tissue engineering scaffolds to improve the regeneration efficiency.