Tooth root development occurs through the interaction of multiple growth factors and transcription factors expressed in Hertwig's epithelial root sheath (HERS) and dental mesenchyme. Previously, we demonstrated that bobby sox homolog (Bbx) regulates odontoblast differentiation of human dental pulp stem cells. Here, we generated Bbx knockout (Bbx−/−) mice to address the functional role of Bbx in tooth formation. During tooth development, Bbx was expressed in both dental epithelium and mesenchyme. However, molar and incisor morphology in Bbx−/− mice at postnatal Day 0 (P0) exhibited no prominent abnormalities compared with their wild‐type (Bbx+/+) littermates. Until P28, the crown morphology in Bbx−/− mice was not distinctively different from Bbx+/+ littermates. Meanwhile, the length of the mandibular base in Bbx−/− mice was notably less at P28. Compared with Bbx+/+ mice, the mesial and distal root lengths of the first molar were reduced by 21.33% and 16.28% at P14 and 16.28% and 16.24% at P28, respectively, in Bbx−/− mice. The second molar of Bbx−/− mice also showed 10.16% and 6.4% reductions at P28 in the mesial and distal lengths, compared with Bbx+/+ mice, respectively. The gene expression analysis during early tooth root formation (P13) showed that the expression of dentin sialophosphoprotein (Dspp) was significantly decreased in Bbx−/− mice. Collectively, our data suggest that Bbx participates in tooth root formation and might be associated with the regulation of Dspp expression. Compared with Bbx+/+ mice, the mesial and distal root lengths of the first and second molars were reduced in Bbx−/− mice. The length of the mandibular base in Bbx−/− mice was notably less at P28. The gene expression analysis during early tooth root formation showed that the expression of Dspp was significantly decreased in Bbx−/− mice. Collectively, our data suggest that Bbx participates in tooth root formation and might be associated with the regulation of Dspp expression. Bbx, bobby sox homolog; Dspp, dentin sialophosphoprotein.