Previous studies have investigated the role of Toll-like receptor (TLR)2 and TLR4 in susceptibility to and severity of Chlamydia trachomatis infections. In this study we employ a unique integrated approach to study the role of the intracellular CpG DNA receptor: we use a murine knockout (KO) model to assess TLR9 relevance, study human TLR9 genotypes and haplotypes in sexually transmitted disease (STD) patients and subfertile women with or without tubal pathology and use in silico TLR9 CpG index calculations to assess potential immunostimulatory properties of the Chlamydia bacterium. Although no significant differences in the course of initial infections were observed between KO mice and wild-type mice the TLR9 KO mice showed a significant level of protection upon reinfection (P = 0.02). We did not observe significant differences in genotype frequencies between C. trachomatis-positive and C. trachomatisnegative women (STD patients). However, haplotype analyses revealed a trend between C. trachomatis-positive and C. trachomatis-negative women in the carriage of haplotype IV (P = 0.061; OR: 2.6; 95% CI: 1.0-6.8). In women with subfertility, odds ratios between 2 and 3 were found for tubal pathology risk, but they did not reach significance due to cohort size limitations. Finally, CpG sequence analysis showed mildly immunostimulatory properties for the genomic sequences of Chlamydia serovars B and D. Based on the murine model, human immunogenetic studies and in silico CpG index analyses, TLR9 seems to play a modest role in C. trachomatis infections. Extension of the human cohorts is necessary to significantly prove the effect in humans.