Human dental pulp stem cells (hDPSCs) are easily obtained multipotent cells, however, their potential value in regenerative medicine is hindered by the phenotypic and functional changes after conventional monolayer expansion. Here, we employed single-cell RNA sequencing (scRNA-seq) to comprehensively study the transcriptional difference between the freshly isolated and monolayer cultured DPSCs. The cell cluster analysis based on our scRNA-seq data showed that monolayer culture resulted in a significant cellular composition switch compared to the freshly isolated DPSCs. However, one subpopulation, characterized as MCAM(+)JAG(+)PDGFRA(?), maintained the most transcriptional characteristics compared to their freshly isolated counterparts. Notably, immunofluorescent staining revealed that the MCAM(+)JAG(+)PDGFRA(?) hDPSCs uniquely located in the perivascular region of human dental pulp tissue. Flow-cytometry analysis confirmed that their proportion remained relatively stable (～2％) regardless of physiological senescence or dental caries. Consistent with the annotation of scRNA-seq data, MCAM(+)JAG(+)PDGFRA (?) hDPSCs showed higher proliferation capacity and enhanced in vitro multilineage differentiation potentials (osteogenic, chondrogenic and adipogenic) compared with their counterparts PDGFRA(+) subpopulation. Furthermore, the MCAM(+)JAG(+) PDGFRA(?) hDPSCs showed enhanced bone tissue formation and adipose tissue formation after 4-week subcutaneous implantation in nude mice. Taken together, our study for the first time revealed the cellular composition switch of monolayer cultured hDPSCs compared to the freshly isolated hDPSCs. After in vitro expansion, the MCAM(+)JAG(+)PDGFRA(?) subpopulation resembled the most transcriptional characteristics of fresh hDPSCs which may be beneficial for further tissue regeneration applications.