The use of three-dimensional porous scaffolds derived from decellularized extracellular matrix (ECM) is increasing for functional repair and regeneration of injured bone tissue. Because these scaffolds retain their native structures and bioactive molecules, in addition to showing low immunogenicity and good biodegradability, they can promote tissue repair and regeneration. Nonetheless, imitating these features in synthetic materials represents a challenging task. Furthermore, due to the complexity of bone tissue, different processes are necessary to maintain these characteristics. We present a novel approach using decellularized ECM material derived from bovine cancellous bone by demineralization, decellularization, and hydrolysis of collagen to obtain a three-dimensional porous scaffold. This study demonstrates that the three-dimensional porous scaffold obtained from bovine bone retained its osteoconductive and osteoinductive properties and presented osteogenic potential when seeded with human Wharton’s jelly mesenchymal stromal cells (hWJ-MSCs). Based on its characteristics, the scaffold described in this work potentially represents a therapeutic strategy for bone repair.