Developmental defects of enamel are common due to genetic and environmental factors before and after birth. Cdc42, a Rho family small GTPase, regulates prenatal tooth development in mice. However, its role in postnatal tooth development, especially enamel formation, remains elusive. Here, we investigated Cdc42 functions in mouse enamel development and tooth repair after birth. Cdc42 showed highly dynamic temporospatial patterns in the developing incisors, with robust expression in ameloblast and odontoblast layers. Strikingly, epithelium-specific Cdc42 deletion resulted in enamel defects in incisors. Ameloblast differentiation was inhibited, and hypomineralization of enamel was observed upon epithelial Cdc42 deletion. Proteomic analysis showed that abnormal mitochondrial components, phosphotransferase activity, and ion channel regulator activity occurred in the Cdc42 mutant dental epithelium. Reactive oxygen species accumulation was detected in the mutant mice, suggesting that abnormal oxidative stress occurred after Cdc42 depletion. Moreover, Cdc42 mutant mice showed delayed tooth repair and generated less calcified enamel. Mitochondrial dysfunction and abnormal oxygen consumption were evidenced by reduced Apool and Timm8a1 expression, increased Atp5j2 levels, and reactive oxygen species overproduction in the mutant repair epithelium. Epithelium-specific Cdc42 deletion attenuated ERK1/2 signaling in the labial cervical loop. Aberrant Sox2 expression in the mutant labial cervical loop after clipping might lead to delayed tooth repair. These findings suggested that mitochondrial dysfunction, up-regulated oxidative stress, and abnormal ion channel activity may be among multiple factors responsible for the observed enamel defects in Cdc42 mutant incisors. Overall, Cdc42 exerts multidimensional and pivotal roles in enamel development and is particularly required for ameloblast differentiation and enamel matrix formation.