BACKGROUND: Human mesenchymal stem cells are being used for various regenerative applications in past decades.This study chronicled a temporal profile of the transcriptional pattern and promoter methylation status of the osteogenic related gene in dental pulp stem cells (DPSCs) derived from 3-dimensional spheroid culture (3D) vis a vis 2-dimensional (2D) monolayer culture upon osteogenic induction. METHODS: Biomimetic properties of osteogenesis were determined by alkaline phosphatase assay and alizarin red staining. Gene expression and promoter methylation status of osteogenic genes such as runt-related transcription factor-2, collagen1α1, osteocalcin (OCN), and DLX5 (distal-homeobox) were performed by qPCR assay and bisulfite sequencing, respectively. Furthermore, μ-Computed tomography (micro-CT) was performed to examine the new bone formation in critical-sized rat calvarial bone defect model. RESULTS: Our results indicated a greater inclination of spheroid culture-derived DPSCs toward osteogenic lineage than the monolayer culture. The bisulfite sequencing of the promoter region of osteogenic genes revealed sustenance of low methylation levels in DPSCs during the progression of osteogenic differentiation. However, the significant difference in the methylation pattern between 2D and 3D derived DPSCs were identified only for OCN gene promoter. We observed differences in the mRNA expression pattern of epigenetic writers such as DNA methyltransferases (DNMTs) and methylcytosine dioxygenases (TET) between the two culture conditions. Further, the DPSC spheroids showed enhanced new bone formation ability in an animal model of bone defect compared to the cells cultivated in a 2D platform which further substantiated our in-vitro observations. CONCLUSION The distinct cellular microenvironment induced changes in DNA methylation pattern and expression of epigenetic regulators such as DNMTs and TETs genes may lead to increase expression of osteogenic markers in 3D spheroid culture of DPSCs which make DPSCs spheroids suitable for osteogenic regeneration compared to monolayers.