Estrogen deficiency-induced osteoporosis typically occurs in postmenopausal women and has been strongly associated with periodontal diseases. Periodontal ligament stem cells (PDLSCs) isolated from the periodontal ligament can differentiate into many types of specialized cells, including osteoblast-like cells that contribute to periodontal tissue repair. The Notch signaling pathway is highly conserved and associated with self-renewal potential and cell-fate determination. Recently, several studies have focused on the relationship between Notch signaling and osteogenic differentiation. However, the precise mechanisms underlying this relationship are largely unknown. We have successfully isolated PDLSCs from both ovariectomized (OVX) and sham-operated rats. Both the mRNA and protein levels of Notch1 and Jagged1 were upregulated when PDLSCs were cultured in osteogenic induction media. Mineralization assays showed decreased calcium deposits in OVX-PDLSCs treated with a γ-secretase inhibitor compared with control cells. Thus Notch signaling is important in maintaining the osteogenic differentiation of PDLSCs in osteoporotic rats, which help in the development of a potential therapeutic strategy for periodontal disease in postmenopausal women.