Human adipose-derived stem cells (hADSCs) and dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to trans-differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the osteoblast differentiation of hADSCs and hDPSCs. First, we investigated characterization of hADSCs and hDPSCs using FACS analysis. Mesenchymal stem cell specific markers, CD44 and CD105, were expressed but not hematopoetic markers, CD45 and CD117 in both of hADSCs and hDPSCs. High-performance thin-layer chromatography analysis showed that increased gangliosides were associated with differentiation of hADSCs and hDPSCs into osteoblasts. RT-PCR analysis confirmed that osteoblast specific genes, ALP, BMP-2, collagen were expressed in differentiated osteoblasts, however, the another osteoblast specific gene, osteocalcin, was not expressed. When hADSCs and hDPSCs were cultured under osteoblast-differentiation conditions, alkaline phosphatase (ALP) activity was increased in comparison to hADSCs and hDPSCs. Furthermore, specifically both ALP activity and ganglioside expression increased more in hDPSCs-derived osteoblasts than hADSCs-derived osteoblasts. These results suggest that gangliosides play a more important role in regulating the osteoblast-differentiation of hDPSCs compared to hADSCs.