The dome of the edible button mushroom (Agaricus bisporus) appears like a paediatric femoral bone head, and the hymenium have large surface area for homing of cells. Considering its structural and chemical composition, we explored the feasibility of Agaricus as a botanical scaffold for bone regeneration. Mushroom being phylo-genetically under fungus species, requires complete decellularization to qualify them as a scaffold for tissue regeneration. The decellularization was done with Sodium Deoxycholate and Nucleases. Potassium iodide was used to disinfect the spores and the scaffolds were cross-linked with staged formalin to mechanically strengthen the scaffold. The residual aldehyde was detoxified with L-Glutamic acid. These scaffolds were made congenial for cell adhesion by coating with nanofibres and preserving them in alcohol. Human Mesenchymal stem cells (hMSCs) were seeded into the mushrooms and induced for osteogenic differentiation. Finally, the seeded scaffolds were validated for osteogenic differentiation. The nano-fiber coated mushroom is a novel 3D scaffold for hMSC differentiation into bone without any threat for fungal infection and in-vitro antigenicity. Further animal studies required are to prove the efficacy as a biological scaffold. Moreover this experimental finding is a revelation to prove that the botanical scaffold can be used for bone regeneration. © 2012 American Scientific Publishers. All rights reserved.