OBJECTIVE: This study aimed to evaluate the role of the chromodomain helicase DNA-binding protein 3 (CHD3) in tooth morphogenesis in Chd3 knockout mice. METHODS: Chd3 knockout mice were generated using the CRISPR-Cas9 method. Mandibular first molars were extracted from the mice and their littermates and morphometrically analyzed. Subsequent histological and immunohistochemical analyses of teeth were performed at each developmental stage. Chd3 knockdown in mesenchymal cells from the dental papilla (mDP) and Hertwig's epithelial root sheath (HERS) was performed by Chd3 shRNA transduction or a control using an adenoviral vector. These effects were examined using cell proliferation assays and quantitative real-time polymerase chain reaction. RESULTS: Narrowing of tooth cervical width was observed in mandibular first molars of Chd3 knockout mice. On postnatal day (PN) 8, the cervical width was narrow before root formation in tooth germs. The number of Ki-67-positive cells decreased in the dental mesenchyme at PN1 and apical papilla at PN8. Chd3 promoted the proliferation of dental mesenchymal cells, but no significant changes were observed in HERS epithelial cells. Chd3 maintained sonic hedgehog (Shh) expression and inhibited that of bone morphogenetic protein (Bmp)4 in dental mesenchymal cells, maintaining Shh and Wnt3a expression and inhibited that of Bmp2 in HERS epithelial cells. CONCLUSION: Chd3 may regulate tooth cervical width during the early growth stage of the apical papilla via Shh, Bmp, and Wnt signaling.