A stem cell-derived mineralized extracellular matrix (ECM) may be a good strategy to endow scaffolds with a bone microenvironment, thus inducing bone regeneration. However, it also faces some challenges, such as limited number of cells, additional wound for autologous cell isolation, and time consumption of cell expansion. In this study, we designed a novel tissue-derived ECM scaffold fabricated by depositing porcine small intestinal submucosa (SIS) ECM on true bone ceramic (TBC), which was followed by mineralization treatment (mSIS/TBC). In vitro, compared with pure TBC, mSIS/TBC promoted cell proliferation, cell viability, and osteoblastic differentiation of the newly seeded rat bone marrow mesenchymal stem cells (BMSCs), and upregulation of the messenger RNA (mRNA) level of osteogenesis-related genes. Western blot assay revealed that mSIS/TBC enhanced osteoblastic differentiation through activation of phosphorylated Smad1/5/8 and phosphorylated extracellular signal-regulated kinase (ERK), as an underlying mechanism. In vivo, in a rat cranial critical size defect model, mSIS/TBC scaffolds induced greater bone formation than pure TBC scaffolds. Meanwhile, a comparative study on the capacity of bone regeneration was also carried out between mSIS/TBC and BMSC-derived ECM deposited on TBC scaffold in vivo and in vitro. The results demonstrated that mSIS/TBC scaffolds acquired a comparable bone regeneration efficacy to that of BMSC-derived ECM deposited on TBC scaffolds. Collectively, our results demonstrated that mSIS/TBC enhanced bone regeneration by supporting cell proliferation and cell viability, and by activating Smad1/5/8 and ERK1/2 signal pathways of BMSC in vitro and in vivo; thus, mSIS/TBC is an excellent alternative to stem cell-derived ECM scaffold for bone regeneration.