Objective and Background Human periodontal ligament mesenchymal stem cells (hPDLMSCs) are reported to be responsible for homeostasis and regeneration of periodontal tissue. Although hPDLMSCs are commonly cultured in monolayers, monolayer cultures have been reported as inferior to 3‐dimensional cultures such as spheroids, which are spherical clusters of cells formed by self‐assembly. The aim of this study was to examine the osteogenic phenotype of spheroids of hPDLMSCs, compared with monolayer cultures of hPDLMSC, in vitro and in vivo. Material and Methods Spheroids were formed using microwell chips that were tagged with polyethylene glycol. Mesenchymal stem cell (MSC) markers in hPDLMSC spheroids were examined by flow cytometer. Real‐time polymerase chain reaction analysis was examined to measure the expressions of stemness markers and osteogenesis‐related genes in monolayer and spheroid‐cultured hPDLMSCs. Immunofluorescence analysis was performed to confirm protein expressions of stemness markers in PDLMSC spheroids. Nodule formation assay, alkaline phosphatase (ALP) activity assay and transplantation assay in a mouse calvarial defect model were performed to confirm the osteogenic potential of hPDLMSC spheroids. To elucidate the mechanism of spheroid culture enhanced osteogenesis in hPDLMSCs with osteoinductive medium (OIM), a small interfering RNA (siRNA) assay targeted with secreted frizzled‐related protein 3 (SFRP3) was examined. The levels of SFRP3 expression in monolayer and spheroid‐cultured hPDLMSCs with OIM were measured by real‐time polymerase chain reaction and western blotting analysis. ALP gene expression and ALP activity were examined in SFRP3‐deficient hPDLMSC spheroids. Results The hPDLMSC spheroids expressed MSC markers, which were similar to hPDLMSCs grown in monolayer cultures. Intriguingly, the protein and mRNA expressions of transcription factors that regulate “stemness” were significantly increased in hPDLMSC spheroids, compared with hPDLMSCs in monolayer cultures. Nodule formation by hPDLMSCs was significantly increased in spheroid cultures grown with OIM, compared with monolayer‐cultured hPDLMSCs. ALP activity and expression of osteogenesis‐related genes were also significantly enhanced in hPDLMSC spheroids, compared with monolayer cultures. Treatment with hPDLMSC spheroids significantly enhanced new bone formation in a murine calvarial defect model, compared with hPDLMSCs in monolayer culture. Finally, to elucidate mechanisms by which spheroid culture enhances ALP activation in hPDLMSCs grown with OIM, an siRNA assay was used to manipulate expression of SFRP3, a Wnt signaling antagonist. Knockdown of SFRP3 suppressed ALP gene expression in hPDLMSCs grown in OIM; further, it suppressed ALP activity in spheroid culture. These data suggest that the enhancement of osteogenic potential in hPDLMSC spheroids is regulated through SFRP3‐mediated ALP activation. Conclusion Spheroid cultures of hPDLMSCs may be a novel and useful tool in regenerative medicine.