Objective: Dental caries is a multifactorial, infectious disease where genetic predisposition plays an important role. Insertion/deletion (I/D) polymorphism of angiotensin-converting enzyme (ACE) has very recently been associated with caries in Polish children. The aim of this study was to analyze ACE I/D polymorphism in a group of caries-free children versus subjects affected by dental caries in the Czech population. Materials and Methods: In this case-control study, 182 caries-free children (with decayed/missing/filled teeth, DMFT = 0), 561 subjects with dental caries (DMFT ≥1) aged 13-15 years and 220 children aged 2-6 years with early childhood caries (ECC, dmft ≥1) were included. Genotype determination of ACE I/D polymorphism in intron 16 was based on the TaqMan method. Results: Although no significant differences in the allele or genotype frequencies between the caries-free children and those affected by dental caries were observed, statistically significant differences between the children with DMFT = 0 and the subgroup of 179 patients with high caries experience (DMFT ≥4; p < 0.01 and p < 0.05, respectively) were detected. The comparison of DD versus II+ID genotype frequencies between the patients with DMFT ≥1 or DMFT ≥4 and healthy children also showed significant differences (31.5% or 35.6% vs. 23.6%, p < 0.05 or p < 0.01, respectively). A gender-based analysis identified a significant difference in the DD versus II+ID genotype frequencies only in girls (p < 0.05). In contrast, no significant association of ACE I/D polymorphism with ECC in young children was found (p > 0.05). Conclusions:ACE I/D polymorphism may be associated with caries in permanent but not primary dentition, especially in girls in the Czech population.