Stem cells from the apical papilla (SCAPs) are promising candidates for regenerative endodontic treatment and tissue regeneration in general. However, harvesting enough cells from the limited apical papilla tissue is difficult, and the cells lose their primary phenotype over many passages. To get over these challenges, we immortalized human SCAPs with lentiviruses overexpressing human telomerase reverse transcriptase (hTERT). Human immortalized SCAPs (hiSCAPs) exhibited long-term proliferative activity without tumorigenic potential. Cells also expressed mesenchymal and progenitor biomarkers and exhibited multiple differentiation potentials. Interestingly, hiSCAPs gained a stronger potential for osteogenic differentiation than the primary cells. To further investigate whether hiSCAPs could become prospective seed cells in bone tissue engineering,in vitroandin vivostudies were performed, and the results indicated that hiSCAPs exhibited strong osteogenic differentiation ability after infection with recombinant adenoviruses expressing BMP9 (AdBMP9). In addition, we revealed that BMP9 could upregulate ALK1 and BMPRII, leading to an increase in phosphorylated Smad1 to induce the osteogenic differentiation of hiSCAPs. These results support the application of hiSCAPs in tissue engineering/regeneration schemes as a stable stem cell source for osteogenic differentiation and biomineralization, which could be further used in stem cell-based clinical therapies.