BackgroundTooth development, as one of the major mineralized tissues in the body, require fine-tuning of mineralization micro-environment. The interaction between dental epithelium and mesenchyme plays a decisive role in this process. With epithelium–mesenchyme dissociation study, we found interesting expression pattern of insulin-like growth factor binding protein 3 (IGFBP3) in response to disruption of dental epithelium–mesenchyme interaction. Its action and related mechanisms as regulator of mineralization micro-environment during tooth development are investigated.ResultsExpressions of osteogenic markers at early stage of tooth development are significantly lower than those at later stage. BMP2 treatment further confirmed a high mineralization micro-environment is disruptive at early stage, but beneficial at later stage of tooth development. In contrast, IGFBP3's expression increased gradually from E14.5, peaked at P5, and decreased afterwards, demonstrating an inverse correlation with osteogenic markers. RNA-Seq and Co-immunoprecipitation showed that IGFBP3 regulates the Wnt/beta-catenin signaling pathway activity by enhancing DKK1 expression and direct protein–protein interaction. The suppression of the mineralization microenvironment effectuated by IGFBP3 could be reversed by the DKK1 inhibitor WAY-262611, further demonstrating that IGFBP3 exerted its influence via DKK1.ConclusionA deeper understanding of tooth development mechanisms is essential for tooth regeneration, which have great implications for dental care. The current study demonstrated that the IGFBP3 expression is regulated in accordance with the needs of the mineralization microenvironment during tooth development, and IGFBP3 exerts its modulating action on osteogenic/odontogenic differentiation of hDPSCs by DKK1-Wnt/ beta-catenin axis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13062-023-00391-9.