Background <p>Tooth development involves interactions between epithelium and mesenchyme conducted via multiple signaling molecules. This study aimed to investigate the roles of Nuclear factor I-C (NFIC) and Sonic hedgehog (SHH) in regulating enamel and dentin formation in mouse incisors.</p> Methods <p><i>Nfic</i> gene knockout (<i>Nfic</i>^{<i>−/−</i>}) and wild-type (<i>Wt</i>) mice were selected at postnatal days (PN) 0.5, 3.5, 5.5, 7.5, 10.5, 15.5, and 21.5 to observe mandibular incisor development by stereomicroscopic observation, hematoxylin–eosin (HE) staining, and immunohistochemistry for SHH. Exogenous SHH protein were injected into the lower incisors of <i>Nfic</i>^{<i>−/−</i>} and <i>Wt</i> mice from postnatal day (PN) 3.5 to PN6.5. The mice injected with physiological saline were used as control. At PN7.5, the lower incisors and mandibles of treated and control mice were dissected under the microscope. Morphological and histological changes were examined by stereomicroscopic observation, HE staining, and immunohistochemistry for dentin sialoprotein (DSP) and amelogenin.</p> Results <p>After PN3.5, mandibular incisor volumes were smaller in <i>Nfic</i>^{<i>−/−</i>} mutant and its enamel and dentin failed to develop. Immunohistochemical staining showed that <i>Shh</i> was expressed in the cervical loop of the mandibular incisors in <i>Wt</i> at PN5.5, but was virtually undetectable in <i>Nfic</i>^{<i>−/−</i>}. The differences of <i>Shh</i> expression between <i>Wt</i> and <i>Nfic</i>^{<i>−/−</i>} were statistically significant both at PN 3.5 and PN5.5 (<i>P</i>&lt;0.05). After injection of exogenous SHH protein into the lower incisors of <i>Nfic</i>^{<i>−/−</i>} mice, enamel and dentin formation was rescued at PN7.5. Immunohistochemistry also confirmed that the expression of DSP and amelogenin was recovered in <i>Nfic</i>^{<i>−/−</i>} mice.</p> Conclusion <p>Enamel and dentin formation in mouse incisors is regulated by NFIC via SHH as a downstream target.</p>