Dental follicle cells (DFCs) are reported to contain stem cells. The canonical Wnt signaling pathway plays an important role in stem cell self-renewal and tooth development through β-catenin expression. The objective of this study was to investigate whether Wnt/β-catenin signaling pathway participates in the cementoblast/osteoblast differentiation of rat DFCs. Immunohistochemistry was used to compare the expression of β-catenin in rat mandibular first molars from postnatal days 1-13. The effects of Wnt/β-catenin signaling on DFCs in vitro were examined by lithium chloride (LiCl) treatment by immunofluorescence, cell counting, dual-luciferase reporter assays, western blotting, and alkaline phosphatase activity analysis. β-Catenin expression was absent in the dental follicles on days 1 and 3 in vivo. It then progressively increased from days 5 to 13. In vitro studies of the DFCs showed that LiCl stimulation caused β-catenin, which was mainly located in the cell membrane and cytoplasm of DFCs, to be immediately transferred to the nucleus and led to the inhibition of proliferation at 12 and 24 hr. LiCl treatment also downregulated the levels of phosphorylated-β-catenin, while upregulating the levels of total β-catenin, nuclear β-catenin, osteocalcin, runt-related transcription factor 2, and collagen type I. In addition, LiCl enhanced the β-catenin/T-cell factor luciferase activity and alkaline phosphatase activity. These results suggest that Wnt/β-catenin signaling pathway positively regulates the cementoblast/osteoblast differentiation of the DFCs.