The existence of PDLSCs [PDL (periodontal ligament) stem cells] in PDL has been identified and such cells may function in periodontal reconstruction, including bone formation. Oestrogens/ERs (oestrogen receptors; ERα and ERβ) exert important effects in bone formation, however, the relationship between ERs and PDLSCs has not been established. In the present study, PDLSCs were isolated and assays for detecting stem-cell biomarkers and multipotential differentiation potential confirmed the validity of human PDLSCs. The results of RT-PCR (reverse transcription-PCR) and Western blotting showed that ERα and ERβ were expressed at higher levels in PDLSCs as compared with PDLCs (PDL cells), and 17β-oestradiol obviously induced the osteogenic differentiation of PDLSCs in vitro. Furthermore, a pan-ER inhibitor or lentivirus-mediated siRNA (small interfering RNA) targeting ERα or ERβ blocked the oestrogen-induced osteogenic differentiation of PDLSCs. The results indicate that both ERα and ERβ were involved in the process of osteogenic differentiation of PDLSCs.