Human adult dental pulp stem cells (hDPSCs) are a unique precursor population isolated from postnatal dental pulp and have the ability to regenerate a reparative dentin‐like complex. In this study, we investigated the role of Asporin in hDPSCs, which was identified as a matrix protein in our previous dentin proteomic analysis. We isolated a clonogenic, highly proliferative population of cells from adult human dental pulp. These isolated hDPSCs were confirmed by fluorescence activated cell sorting (FACS) using stem cell‐specific markers and have shown multilineage differentiation potential. The localization of Asporin was identified by immunohistochemistry in the globular calcification region in the junction of predentin and dentin. The gene and protein expression levels of Asporin were enhanced at the early stage of and then reduced during the late stage of differentiation of hDPSCs in mineralization media. ASPN knock‐down using a lentiviral system suppressed the mineralization of hDPSCs. These results suggest that ASPN plays positive roles in the mineralization of hDPSCs and predentin to dentin. J. Cell. Physiol. 226: 1676–1682, 2011. © 2010 Wiley‐Liss, Inc.