HPDLSCs derived from periodontal ligament tissues contribute to tooth development and tissue regeneration. Exploring the effects of long noncoding RNAs (lncRNAs) in the process of osteogenic differentiation of periodontal ligament stem cells would provide novel therapeutic strategies for tissue regeneration. The expression levels of lncRNA, which significantly changed during osteogenic differentiation, were observed by real-time quantitative PCR (q-PCR). Then, we screened for osteogenic-related lncRNA, which was initially named lncRNA-TWIST1. Moreover, we detected the mRNA expression levels of TWIST1 and osteogenesis-related genes after upregulating and downregulating lncRNA-TWIST1 in PPDLSCs (periodontal mesenchymal stem cells from periodontitis patients) and HPDLSCs (periodontal mesenchymal stem cells from healthy microenvironment), respectively. The osteogenic degree was verified by detecting ALP activity and alizarin red staining. LncRNA-TWIST1 decreased the mRNA levels of TWIST1 and promoted osteogenic differentiation in PPDLSCs, which was confirmed by the increase in osteogenesis-related gene levels (Runx2, ALP, and OCN), the increase in ALP activity, and the formation of more osteogenic nodules. In contrast, downregulating lncRNA-TWIST1 decreased the expression of osteogenesis-related genes, ALP activity, and osteogenic nodules both in PPDLSCs and in HPDLSCs. LncRNA-TWIST1 promoted osteogenic differentiation both in PPDLSCs and in HPDLSCs by inhibiting the TWIST1 expression. LncRNA-TWIST1 may be a novel therapeutic strategy to regenerate dental tissues.