Nestin is an intermediate filament protein and a marker of odontoblasts, but its function in tooth and bone formation is largely unknown. This study aimed to determine whether Nestin plays a role in postnatal tooth formation. 4-week-old Nestin knockout (KO) mice were analyzed with a range of techniques, including X-ray imaging, uCT, backscattered and acid-etched casted SEM, FITC-confocal microscopy, H&E and TRAP staining, and immunohistochemistry. The KO mice had no apparent difference in crown formation compared to age-matched wild-type (WT) but showed delayed molar eruption with reduced TRAP osteoclasts. More importantly, KO mice developed expanded predentin and shorter, thinner roots with irregular and shortened dentin tubules. Additionally, the Nestin KO mice exhibited a reduced cellular cementum mass with sharp reductions in DMP1, OPN, and BSP. These findings suggest that Nestin plays a critical role in the postnatal development of root dentin and cellular cementum.