Human periodontal ligament stem cells (hPDLSCs) are mesenchymal stem cells (MSCs) derived from dental and craniofacial tissues that exhibit high potential for differentiation into osteoblasts. Recently, microRNAs (miRNAs) have been established to play important roles in MSC osteogenesis. In the current study, we report that miR-21 was down-regulated in osteogenically differentiated PDLSCs. Overexpression of miR-21 significantly inhibited osteogenesis of hPDLSC, whereas its inhibition demonstrated the opposite effects. Furthermore, SMAD family member 5 (Smad5) was predicted to be a downstream target of miR-21 and was shown to undergo up-regulation in PDLSCs induced toward osteogenesis. Moreover, Smad5 and Runx2, which are the critical transcription factors in osteogenic differentiation, were predicted to be targets of miR-21. Suppression of miR-21 expression increased the level of Smad5 in vitro and during in vivo transplantation experiments. Furthermore, suppression of Smad5 inhibited osteogenic differentiation and decreased the protein level of Runx2. Taken together, these results suggested that miR-21 be mechanistically implicated in the regulation of osteogenic differentiation of hPDLSCs by targeting Smad5.