The purpose of this study was to investigate the effect of sodium butyrate (SB)-containing calcium sulfate (CaS) bone graft on fibroblasts, oral mucosa and bone tissue. All the tests were performed according to the standard method of ISO 10993. For the cytotoxicity assay, the SB/CaS mixture was set for 24 h, and was placed on the layer of fibroblasts covered with agar for 24 h. Most cells under and near the mixture were viable and showed the morphology of healthy cells, which indicated that there was no cytotoxicity. The effect of SB/CaS mixture on oral mucosa was evaluated using the hamster cheek pouch. There were no signs of tissue responses indicating inflammatory reactions to SB/CaS mixture. Finally, there was no appearance of inflammatory cells, and normal tissue histology was shown by the implantation of SB/CaS mixture to the femur of rabbits. Therefore, it was considered that the SB/CaS mixture was non-cytotoxic and non-irritant to oral mucosa and bone tissue.