The development of periodontal ligament-cementum complex (PLCC) originates from the interaction between epithelial cells of Hertwig's epithelial root sheath (HERS) and mesenchymal cells of the dental follicle. While previous studies have suggested that the Wnt pathway is involved in osteogenic differentiation of dental follicle cells (DFCs) during tooth root development, its involvement in the interaction between DFCs and HERS cells (HERSCs) in tooth root mineralization remains unclear. Here, we investigated the hypothesis that HERSCs control osteogenic differentiation of DFCs via the Wnt pathway. We found that during co-culture with HERSCs, DFCs exhibited a greater tendency to form mineralized nodules. Moreover, under these conditions, DFCs expressed high levels of cementoblast/osteoblast differentiation-related markers, such as bone sialoprotein (BSP) and osteocalcin (OCN), the periodontal ligament phenotype-related gene type I collagen (COL1), and β-catenin (CTNNB1), a core player in the canonical Wnt pathway. In contrast, expression in DFCs of alkaline phosphatase (ALP) was greatly decreased in the presence of HERSCs. Expression of CTNNB1 in DFCs was stimulated by Wnt3a, a representative canonical member of the Wnt family of ligands, but suppressed by Dickkopf1 (DKK1), a Wnt/CTNNB1 signaling inhibitor. Furthermore, in the presence of treated dentin matrix (TDM), differentiation of DFCs was enhanced by Wnt3a when they were in direct contact with HERSCs, but was curtailed by DKK1. Taken together, these results indicate that during tooth root formation, HERSCs induce osteogenic differentiation of DFCs in a process involving the Wnt pathway and the dentin matrix. Our study not only contributes to our understanding of tooth root development and diseases of tooth root mineralization, but also proffers a novel potential strategy for controlling mineralization during tooth root regeneration.