Mineral trioxide aggregate (MTA) has recently been used as a pulpotomy medicament for primary molars. The aim of this study was to evaluate and compare the proliferation and differentiation potential of dental pulp stromal cells of permanent teeth and deciduous teeth cultured on MTA-coated surface. Human dental pulp stromal cells were obtained from human permanent premolars and deciduous teeth and cultured on MTA-coated culture plates. The cells were subjected to proliferation assay and cell cycle analysis. Their differentiation potential was evaluated by analysing changes in the mRNA expressions of runt-related transcriptional factor 2 (Runx2) and alkaline phosphatase (ALP). Morphological changes of cells in direct contact with MTA were observed using scanning electron microscopy (SEM). The proliferation rates, distribution of cell cycles and mRNA expression patterns of Runx2 and ALP were similar in both types of pulpal cells. SEM observations revealed that both types changed into more dendrite-like cells. On the surface of MTA, human dental pulp stromal cells from deciduous and permanent teeth were able to both proliferate and differentiate into cells that induce mineralization. MTA is suitable as a biocompatible pulpotomy medicament for primary teeth.