Conventional root canal treatment may result in loss of tooth vitality, which can lead to unfavorable treatment outcomes. Notably, a ceased tooth development of immature permanent teeth with open apices, regeneration of periodontal ligaments (PDL), and pulp is highly expected healing process. For regeneration, the scaffold is one of the critical components that carry biological benefits. Therefore, this study evaluated a decellularized human tooth as a scaffold for the PDL and pulp tissue regeneration. A tooth scaffold was fabricated using an effective decellularization method as reported in previous studies. PDL stem cells (PDLSCs) and dental pulp stem cells (DPSCs) obtained from human permanent teeth were inoculated onto decellularized scaffolds, then cultured to transplant into immunosuppressed mouse. After 9 weeks, PDLSCs and DPSCs that were inoculated onto decellularized tooth scaffolds and cultured in an in vivo demonstrated successful differentiation. In PDLSCs, a regeneration of the cementum/PDL complex could be expected. In DPSCs, the expression of genes related to revascularization and the hard tissue regeneration showed the possibility of pulp regeneration. This study suggested that the potential possible application of decellularized human tooth could be a scaffold in regeneration PDL and pulp tissue along with PDLSCs and DPSCs, respectively, as a novel treatment method.