Human periodontal ligament stem cells (hPDLSCs) are promising seed cells for oral bone tissue engineering. Dermatopontin (DPT) is a small-molecule protein recognized as a non-collagenous component of the extracellular matrix and is associated with a variety of biological processes. In this study, we first determined that DPT was elevated during the osteogenic differentiation of hPDLSCs. HPDLSCs interfering with DPT expression were established by lentiviral infection. It was found that the proliferation and osteogenic differentiation ability of hPDLSCs were inhibited after interfering DPT with lentivirus. Exogenous recombinant DPT treatment could not alter the proliferation of hPDLSCs. Coincidentally, exogenous DPT can only enhance the osteogenic differentiation of hPDLSCs in the control lentivirus group, but had no significant effect on the DPT interference group. This study expands the understanding of DPT function and implicates DPT as an important target for enhancing osteogenic differentiation of hPDLSCs.