Bone autografts are often used for reconstruction of bone defects; however, due to the limitations of autografts, researchers have been in search of bone substitutes. Dentin is of particular interest for this purpose due to high similarity to bone. This in vitro study sought to assess the surface characteristics and biological properties of dentin samples prepared with different treatments. This study was conducted on regular (RD), demineralized (DemD), and deproteinized (DepD) dentin samples. X-ray diffraction and Fourier transform infrared spectroscopy were used for surface characterization. Samples were immersed in simulated body fluid, and their bioactivity was evaluated under a scanning electron microscope. The methyl thiazol tetrazolium assay, scanning electron microscope analysis and quantitative real-time polymerase chain reaction were performed, respectively to assess viability/proliferation, adhesion/morphology and osteoblast differentiation of cultured human dental pulp stem cells on dentin powders. Of the three dentin samples, DepD showed the highest and RD showed the lowest rate of formation and deposition of hydroxyapatite crystals. Although, the difference in superficial apatite was not significant among samples, functional groups on the surface, however, were more distinct on DepD. At four weeks, hydroxyapatite deposits were noted as needle-shaped accumulations on DemD sample and numerous hexagonal HA deposit masses were seen, covering the surface of DepD. The methyl thiazol tetrazolium, scanning electron microscope, and quantitative real-time polymerase chain reaction analyses during the 10-day cell culture on dentin powders showed the highest cell adhesion and viability and rapid differentiation in DepD. Based on the parameters evaluated in this in vitro study, DepD showed high rate of formation/deposition of hydroxyapatite crystals and adhesion/viability/osteogenic differentiation of human dental pulp stem cells, which may support its osteoinductive/osteoconductive potential for bone regeneration.