Abstract Introduction Odontoblasts are terminally differentiated cells of ectomesenchymal origin that produce the dentin. Differentiated odontoblasts cannot be identified yet by a single phenotypic marker protein; therefore, a combination of markers is currently used. Up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 has been associated with exit from the cell cycle and terminal differentiation of mammalian cells. Immunoreactivity for p27Kip1 protein was shown in many adult mouse tissues, but no information is available on the expression of p27Kip1 in mammalian dental pulp. Methods Healthy and carious adult human molars with reparative dentin formation were decalcified, cryoprotected, frozen embedded, and frozen sectioned. The expression of p27Kip1 and nestin in cells of adult human dental pulp was analyzed by immunohistochemistry using free floating sections. Results p27Kip1 showed strong nuclear expression in many differentiated human molar odontoblasts at the odontoblastic layer. Most cells of the cell-rich zone displayed low levels of p27Kip1 despite the fact that preodontoblasts localized in the cell-rich zone of the subodontoblastic layer have been identified as quiescent cells. The nuclear expression of p27Kip1 in stromal cells of the dental pulp was variable, indicating that subpopulations of these cells were in distinct states of differentiation. Odontoblasts generating reparative dentin showed comparable nuclear expression of p27Kip1 in comparison with odontoblasts synthesizing primary/secondary dentin. This result indicates that odontoblasts synthesizing primary/secondary or reparative dentin exhibit a similar differentiation status. Conclusions Our findings show that increased expression of nuclear p27Kip1 occurred during differentiation from preodontoblasts to odontoblasts in adult healthy and carious molars. p27Kip1 can be used as a novel nuclear marker protein for differentiated human odontoblasts in vivo.