Tooth root morphogenesis involves two biological processes, root elongation and dentinogenesis, which are guaranteed by downgrowth of Hertwig's epithelial root sheath (HERS) and normal odontoblast differentiation. Ubiquitin-dependent protein degradation has been reported to precisely regulate various physiological processes, while its role in tooth development is still elusive. Here we show ubiquitin-specific protease 34 (USP34) plays a pivotal role in root formation. Deletion of Usp34 in dental mesenchymal cells leads to short root anomaly, characterized by truncated roots and thin root dentin. The USP34-deficient dental pulp cells (DPCs) exhibit decreased odontogenic differentiation with downregulation of nuclear factor I/C (NFIC). Overexpression of NFIC partially restores the impaired odontogenic potential of DPCs. These findings indicate that USP34-dependent deubiquitination is critical for root morphogenesis by stabilizing NFIC.