Objective: To investigate the role of microbiota in dentine formation and the characteristics of dental pulp stem cells (DPSCs) in mouse incisors. Methods: The influence of microbiota on dentine was detected via microcomputed tomography (microCT), microhardness testing and haematoxylin-eosin (HE) staining in incisors from germ-free (GF), specific pathogen–free (SPF) and conventionalised (ConvD) mice. Cell Counting Kit-8 (CCK-8) assay, alizarin red staining and expression of dentine sialophosphoprotein (DSPP), alkaline phosphatase (ALP) and bone sialoprotein (BSP) via real-time polymerase chain reaction (PCR) were used to evaluate the biological characteristics of DPSCs derived from mice of different microbiota status. Results: MicroCT showed that the incisors in the GF and ConvD groups had comparable dentine thickness to those in the SPF group. Microhardness testing showed a lower dentine hardness value in GF incisors compared to SPF, while HE staining showed that GF incisors exhibited thicker predentine than SPF incisors. There was no difference between the ConvD and SPF groups. DPSCs from GF mice showed no significant difference in proliferation rate to SPF and ConvD DPSCs. DPSCs from GF mice formed less mineral deposition and expressed lower levels of osteo-/odontogenic differentiation–related genes including ALP, BSP and DSPP than SPF and ConvD DPSCs. The absence of microbiota in GF mice resulted in a lower dentine hardness value, thicker predentine and impaired osteo-/odontogenic differentiation capacity. Conclusion: The absence of microbiota impaired the dentine mineralisation and osteo-/odontogenic differentiation abilities of DPSCs. Keywords: dental pulp stem cells, dentinogenesis, germ-free, microbiota, mineralisation