Objective To investigate the effects of vacuolar protein sorting 4B ( VPS4B) on odontogenic differentiation and Notch pathway signaling in human dental pulp stem cells ( hDPSCs). Methods hDPSCs was divided into normal, mineralized, negative control, and VPS4B-siRNA groups. The normal group was grown in normal culture, without any additional treatment. The remaining groups underwent treatment with mineralized liquid ( 0. 785 g / L dexamethasone, 0. 05 g / L vitamins, 2. 16 g / L sodium β-glycerophosphate); the negative control and VPS4B-siRNA groups underwent transfection of negative control siRNA and VPS4B-siRNA, respectively, using Lipofectamine 2000. The CCK8 assay was used to detect cell proliferation. The protein levels of VPS4B, Notch intracellular domain (NICD), and hairy and enhancer of split-related with YRPW motif1 (Hey1) were detected by Western blotting. Cell mineralization was observed by alizarin red staining. Cell calcium concentrations were determined using a calcium detection kit. The mRNA levels of Runx2, osteopontin (OPN), and dentin sialophosphoprotein (DSPP) were detected by real-time fluorescence quantitative PCR. Results In the normal group, the alizarin red staining area was light and small. The mineralized and negative control groups exhibited a darker and larger staining area, while the VPS4B-siRNA group exhibited a lighter and smaller staining area. Compared with the normal group, the levels of OD₄₅₀ , NICD, and Hey1 proteins were reduced in the mineralized and negative control groups (all P< 0. 05), while the relative number of mineralized nodules, level of VPS4B protein, concentration of calcium, and levels of Runx2, OPN, and DSPP mRNA were enhanced ( all P< 0. 05). The relative number of mineralized nodules and levels of OPN and DSPP mRNA were enhanced in the VPS4B-siRNA group (all P< 0. 05), while the levels of OD₄₅₀ , NICD, and Hey1 proteins were reduced ( all P< 0. 05). Compared with the mineralized and negative control groups, NICD and Hey1 protein levels were enhanced in the VPS4B-siRNA group (both P< 0. 05), while the levels of OD₄₅₀ and VPS4B protein, the relative number of mineralized nodules, concentration of calcium, and levels of Runx2, OPN, and DSPP mRNA were reduced ( all P< 0. 05). Conclusions Interference with VPS4B activity can inhibit odontogenic differentiation in hDPSCs, presumably through modulation of the Notch pathway.