Abstract： Objective:To explore the effect of cell division cycle 42 (CDC42) on root development and its regulation on cell proliferation and migration in Hertwig root sheath (HERS).Methods:Trace the spatiotemporal expression of CDC42 in root development process [postnatal day 5 (P5), P7, P14] through immunofluorescence staining. Nine eight-week-old C57BL/6J male mice were randomly divided into 3 groups using a simple random sample method ( n=3 in each group). P3 tooth germ was cultured in air-liquid system for 1 day and then transplanted to renal capsule each to observe tooth root development. The control group implanted tooth germ only. The phosphate buffered saline (PBS) group implanted tooth germ and gel beads soaked with PBS, while the ML141 group implanted tooth germ and gel beads soaked with CDC42 inhibitor (ML141). Cdc42 in HERS cells was inhibited via lentivirus transfection. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2′-deoxyuridine (EdU) assay and scratch assay were performed. The distribution of Golgi apparatus (GM130) and cytoskeleton (F-actin) in migrated cells were mapped via immunofluorescence staining. Results:CDC42 was expressed in epithelial cells of HERS, polarized ameloblasts and odontoblasts, as well as adjacent dental papilla and dental follicle cells during tooth root development. The root length of the ML141 group [(0.61±0.09) mm] was substantially shorter than that of control group [(1.03±0.19) mm, P=0.007] and PBS group [(0.98±0.10) mm, P=0.021] according to the data of renal capsule transplantation. After lentiviral transfection, the relative expression of Cdc42 in knockdown group (0.31±0.33) was significantly lower than that in control group (1.05±0.08) ( t=15.38, P<0.001), demonstrating the knockdown efficiency closed to 70%. Cell viabilities were significantly inhibited in knockdown group (0.87±0.04, 0.96±0.10, 0.59±0.06, respectively) compared with those in control group (1.09±0.13, 1.55±0.32, 1.10±0.09, respectively) after 3, 4 and 5 days ( t=3.16, P=0.016; t=4.23, P=0.002; t=5.08, P<0.01), and the cell proliferation ability in knockdown group [(1.65±0.64)%] also decreased than that in the control group [(4.02±1.12)%]( t=5.21, P<0.001). In addition, the cell migration rates after 24 and 48 h [(45.1±4.2)%, (56.4±8.3)%] in knockdown group were obviously lower than those in the control group [(63.8±7.4)%, (80.2±7.8)%] ( t=3.78, P=0.019; t=3.62, P=0.023). After Cdc42 was knocked down, Golgi apparatus distributed along the nucleus while behaved oriented in the control group. Conclusions:CDC42 plays an important role in the regulation of root length during root development, which may mediate root elongation by affecting the migration and proliferation of HERS cells.