Recently, it has been reported that the orphan nuclear receptor estrogen-related receptor α (ERRα) is involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Moreover, ERRα has been identified as a novel therapeutic target for treating osteoporosis and other bone diseases. Human periodontal ligament tissue-derived mesenchymal stem cells (hPDLSCs) have recently been used in stem cell-mediated therapies because of their multipotency, particularly toward osteogenic differentiation. However, it is still unclear whether ERRα can regulate the osteogenic differentiation of hPDLSCs. In the present study, we investigated the role of ERRα in the osteogenic differentiation of hPDLSCs in vitro. We isolated hPDLSCs and confirmed their capacity for multipotent differentiation. Furthermore, we examined ERRα expression in hPDLSCs by RT-PCR and immunocytochemistry. We found that the expression of ERRα mRNA was significantly increased during the late stage of osteogenic differentiation of hPDLSCs. Moreover, transfection of recombinant lentiviral-mediated miRNA targeting ERRα significantly suppressed ALP activity, mineralization capacity, and the mRNA expression of osteogenesis-related genes (ALP, OCN, RUNX2 and OPN) in hPDLSCs. Our results indicate that ERRα may promote the osteogenic differentiation of hPDLSCs in vitro.