AbstractDental pulp stem cells (DPSCs) are clonogenic, self‐renewing, and multi‐potential DPSCs capable of differentiating into osteoblasts. In this study, primary cell cultures were obtained from human dental pulp tissue of developing third molars, and flow cytometry was used to sort the subpopulation of DPSCs with STRO‐1 and CD146 double‐positive expression (denoted “DPSCs”). It was noted that DPSCs exhibited superior clonogenic potential and osteogenic differentiation capability than the dental pulp cell subpopulation with STRO‐1 and CD146 double‐negative expression (denoted DPCs). Furthermore, a low concentration (0.005 mg/ml) of exogenous glucosamine (GlcN) was effective in promoting the early osteogenic differentiation of DPSCs through the transforming growth factor‐β receptor (TGF‐βr) type I and Smads signal pathways, which upregulated the Runt‐related transcription factor 2/core‐binding factor alpha1 (Runx2/Cbfa1) and alkaline phosphatase at both the mRNA and protein levels. In the presence of osteogenic supplements, GlcN‐treated DPSCs produced more mineralized‐matrix deposition than did the untreated groups. Taken together, this study demonstrates the capacity of GlcN to promote the osteogenic differentiation of human DPSCs, and the underlying mechanism involves a TGF‐βr‐dependent Smad signal pathway. J. Cell. Physiol. 225: 140–151, 2010. © 2010 Wiley‐Liss, Inc.