Since bone defects can lead to various disabilities, in recent years, many increasing attempts have been made in bone tissue engineering. In this regard, scaffolds have attracted a lot of attention as three dimensional substrates for cell attachment which improve successful tissue engineering. The aim of the present study was to provide an interconnected porous scaffold to facilitate cell infiltration. To do so, cancellous bone from bovine femur was dissected in fragments and decellularized by physicochemical methods, including snap freeze/thaw, rinsing in hot water and treatment with different solutions of sodium dodecyl sulfate (SDS). Histological analysis and 4',6-diamidino-2-phenylindole staining revealed that the best results were obtained after treatment with 2.5%, 5%, and 8% SDS for 8, 3, or 1 h respectively, which significantly removed bone cells with intact trabeculae geometry. Further characterization of decellularized scaffolds by the compression tests also revealed no significant difference between elastic modulus values of the three different SDS treatments. Moreover, studying the ratio of bone trabeculae to bone surfaces (BT/BS) as assessed by Clemex vision software 3.5 showed that treatment with 2.5% SDS for 8 h resulted in a BT/BS score in the range of native bone and therefore this treatment was used for further experiments. Histological studies and scanning electron microscopy revealed rat mesenchymal stem cells integration, adhesion, and maintenance during the 2 and 7 d of culture in vitro. In conclusion, the present results support the effective role of SDS in cancellous bovine bone decellularization and also propensity of treated samples in providing a suitable three-dimentional environment to support the maintenance and growth of mesenchymal stem cells.