Transgenic mice that express dominant-negative RhoA (RhoADN) in ameloblasts have hypoplastic enamel with defects in molar cusps. β-catenin and Wnt5a were up-regulated in enamel organs of RhoADN transgenic mice, which indicated that both canonical and non-canonical Wnt pathways are implicated in the process of enamel defect formation. It was hypothesized that expression of RhoADN in ameloblasts interfered with normal enamel development through the pathways that were induced by fluoride. The Wnt and RhoA pathways were further investigated in an ameloblast-lineage cell line (ALC) by treatment with sodium fluoride (NaF). The activities of RhoA and Rho-associated protein kinase (ROCK) II decreased significantly by 8–12 hours, similar to decreased activity in RhoADN transgenic mice. Both canonical and non-canonical Wnt pathways were activated by treatment with NaF, which was verified by western blotting and the β-catenin-TCF/LEF (T cell factor lymphanoid/enhancer factor) reporter gene (TOPflash) assay. β-catenin localization to both cytoplasm and nucleus was up-regulated in NaF-treated ALC, while Gsk-3β, the negative regulator of the Wnt pathway, showed a decreased pattern of expression. The current results indicate that both Wnt and RhoA pathways are implicated in fluoride-induced signaling transductions in the ALC as well as in the development of enamel defects in RhoADN transgenic mice.