The evolutionarily conserved Notch signalling pathway regulates the fate, proliferation and differentiation of cells in most developing organs, thus affecting their morphogenesis and function. Here, we investigated the role of the Notch2 receptor in the generation and function of epithelial cells of the continuously erupting rodent incisors. We used transgenic -Cre;Rosa26 and -Cre;Rosa26 mice to compare the contribution of and expressing cells and their progeny in the generation of the different epithelial cell populations. Furthermore, we examined if the dental epithelium organisation and enamel structure are affected in early postnatal incisors of ; mice using immunofluorescent staining, gene expression analysis, microcomputed tomography and scanning electron microscopy. Our results showed that Notch2 deletion resulted in smaller incisors with disorganised dental epithelium and defective enamel. Delayed eruption was correlated with alterations in the proliferative and differentiation status of epithelial stem cells in the cervical loop area of the incisors. Similar results were obtained with in vitro studies, where inhibition of the Notch signalling by the CB103 blocker recapitulated the in vivo phenotype. In conclusion, this study demonstrates for the first time the importance of Notch2 in epithelial cell fate acquisition, dental epithelium organisation and enamel structure in rodent incisors.