Background Periodontitis, a common inflammatory disease in adults, causes the loss of alveolar bone, eventually leading to tooth loss. The inhibition of osteogenic differentiation of periodontal ligament stem cells (PDLSCs) might be involved in the development of alveolar bone loss. Objective The aim of the present study was to investigate the regulatory roles of nuclear factor erythroid-2-related factor 2 (Nrf2) during the osteogenic differentiation of PDLSCs in the presence of LPS. Results In the present study, LPS reduced cell viability, inhibited osteogenic differentiation, and caused oxidative stress in PDLSCs. Antioxidants, glutathione (GSH), N-acetyl-L-cysteine (NAC), and ascorbic acid (AA), had rescue effects. Furthermore, LPS reduced antioxidant capability and expression of Nrf2 and its down-stream antioxidant genes. Nrf2 overexpression abolished the cytotoxic effects of LPS by alleviating ROS level. Conclusion LPS induced oxidative stress through downregulating Nrf2-mediated antioxidant pathways, and inhibited osteogenic differentiation in PDLSCs. Strategies enhancing antioxidant capability could reverse the impaired osteogenic potential caused by LPS.