Tooth agenesis is one of the most common anomalies of human dentition. Recent studies suggest that a number of genes are related to both syndromic and non-syndromic forms of hypodontia. In a previous study, we observed that polymorphism in rs929387 of GLI3 might be associated with hypodontia in the Chinese Han population based on a limited population. To further confirm this observation, in this study, we employed 89 individuals diagnosed with sporadic non-syndromic oligodontia (40 males and 49 females) to investigate the relationship between polymorphism in rs929387 of GLI3 and tooth agenesis. These individuals were analyzed with 273 subjects (125 males and 148 females) diagnosed with non-syndromic hypodontia and 200 healthy control subjects (100 males and 100 females). DNA was obtained from whole blood or saliva samples and genotyping was performed by a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) method. Significant differences were observed in the allele and genotype frequencies of rs929387 of GLI3. Distributions of genotypes TT, TC and CC of rs929387 polymorphism were significantly different between the case group and the control group (P = 0.013) and C allelic frequency was higher in case group [P = 0.002, OR = 1.690, 95% CI (1.200-2.379)]. Additionally, our analysis shows that this difference is more pronounced when compared between the male case group and the male control group. The function study suggests that variation in GLI3 caused by rs929387 leads to a decrease in its transcriptional activity. These data demonstrated an association between rs929387 of GLI3 and non-syndromic tooth agenesis in Chinese Han individuals. This information may provide further understanding of the molecular mechanisms of tooth agenesis. Furthermore, GLI3 can be regarded as a marker gene for the risk of tooth agenesis.