The tooth has a unique configuration with respect to biomaterials that are used for its treatment. Cells inside of the dental pulp interface indirectly with biomaterials via a calcified permeable membrane, formed by the dentin matrix and several thousands of dentinal tubules (∼2 μm in diameter). Although the cytotoxic response of the dental pulp to biomaterials has been extensively studied, there is a shortage of in vitro model systems that mimic the dentin–pulp interface and enable an improved understanding of the morphologic, metabolic and functional influence of biomaterials on live dental pulp cells. To address this shortage, here we developed an organ-on-a-chip model system which integrates cells cultured directly on a dentin wall within a microfluidic device that replicates some of the architecture and dynamics of the dentin–pulp interface. The tooth-on-a-chip is made out of molded polydimethylsiloxane (PDMS) with a design consisting of two chambers separated by a dentin fragment. To characterize pulp cell responses to dental materials on-chip, stem cells from the apical papilla (SCAPs) were cultured in odontogenic medium and seeded onto the dentin surface, and observed using live-cell microscopy. Next, to evaluate the tooth-on-a-chip as a platform for materials testing, standard dental materials used clinically (2-hydroxyethylmethacrylate – HEMA, phosphoric acid – PA, and Adper-Scotchbond – SB) were tested for cytotoxicity, cell morphology, and metabolic activity on-chip, and compared against standardized off-chip controls. All dental materials had cytotoxic effects in both on-chip and off-chip systems in the following order: HEMA > SB > PA (p < 0.05), and cells presented consistently higher metabolic activity on-chip than off-chip (p < 0.05). Furthermore, the tooth-on-a-chip enabled real-time tracking of gelatinolytic activity in a model hybrid layer (HL) formed in the microdevice, which suggests that dental pulp cells may contribute to the proteolytic activity in the HL more than endogenous proteases. In conclusion, the tooth-on-a-chip is a novel platform that replicates near-physiologic conditions of the pulp–dentin interface and enables live-cell imaging to study dental pulp cell response to biomaterials.