Estrogen deficiency in post-menopausal women is considered as one of the risk factors for periodontal diseases. The periodontal ligament is a connective tissue that connects cementum and alveolar bone to constrain teeth within the jaw. Periodontal ligament stem cells (PDLSCs) isolated from the periodontal ligament can differentiate into many types of specialized cells, including osteoblast-like cells that can be used to regenerate alveolar bone. However, little is known about the effect of estrogen-deficient microenvironment on the osteogenic differentiation of PDLSCs. The aim of this study was to explore the role of estrogen on the potential for osteogenic differentiation of PDLSCs using a rat model of osteoporosis. Three-month-old female Sprague-Dawley rats were divided into two groups (n = 6 for each): ovariectomized (OVX) and sham-operated rats (Sham). Then the characteristics of PDLSCs isolated from these rats were investigated. Real-time PCR analysis showed the lower expression levels of estrogen receptors (ERα and ERβ) mRNAs in PDLSCs of OVX animals compared to Sham control. Mineralization assay demonstrated fewer calcium deposits in PDLSCs from OVX group than those from Sham group. Treatment with 17β-estradiol (E2) significantly enhanced the osteogenic differentiation of PDLSCs from both groups in vitro. Furthermore, by means of lentivirus-mediated siRNA targeting ERα or ERβ, the expression of ERα or ERβ was down-regulated (> 50% reduction), which impaired the estrogen-induced osteogenic differentiation of PDLSCs from both groups (> 50% reduction). These results indicate that estrogen plays an important role in maintaining osteogenic differentiation of PDLSCs, which acts through both ERα and ERβ.