Objective To observe the histological structure and cytocompatibility of novel acellular bone matrix (ACBM) and to investigate the feasibility as a scaffold for bone tissue engineering. Methods Cancellous bone columns were harvested from the density region of 18-24 months old male canine femoral head, then were dealt with high-pressure water washing, degreasing, and decellularization with Trixon X-100 and sodium deoxycholate to prepare the ACBM scaffold. The scaffolds were observed by scanning electron microscope (SEM); HE staining, Hoechst 33258 staining, and sirius red staining were used for histological analysis. Bone marrow mesenchymal stem cells (BMSCs) from canine were isolated and cultured with density gradient centrifugation; the 3rd passage BMSCs were seeded onto the scaffold. MTT test was done to assess the cytotoxicity of the scaffolds. The proliferation and differentiation of the cells on the scaffold were observed by inverted microscope, SEM, and live/dead cell staining method. Results HE staining and Hoechst 33258 staining showed that there was no cell fragments in the scaffolds; sirius red staining showed that the ACBM scaffold was stained crimson or red and yellow alternating. SEM observation revealed a three dimensional interconnected porous structure, which was the microstructure of normal cancellous bone. Cytotoxicity testing with MTT revealed no significant difference in absorbance (A) values between different extracts (25%, 50%, and 100%) and H-DMEM culture media (P gt; 0.05), indicating no cytotoxic effect of the scaffold on BMSCs. Inverted microscope, SEM, and histological analysis showed that three dimensional interconnected porous structure of the scaffold supported the proliferation and attachment of BMSCs, which secreted abundant extracellular matrices. Live/dead cell staining results of cell-scaffold composites revealed that the cells displaying green fluorescence were observed. Conclusion Novel ACBM scaffold can be used as an alternative cell-carrier for bone tissue engineering because of thoroughly decellularization, good mircostructure, non-toxicity, and good cytocompatibility.