Background: Chlamydia trachomatis (CT) is one of the most prevalent sexually transmitted infections, causing genital tract infections and infertility. Defensins have an immunomodulatory function and play an important role in sperm maturation, motility, and fertilization. DEFB126 is present on ejaculated spermatozoa and is essential for them to pass through the female reproductive tract. The purpose of the current study was to determine the frequency of the 2-nt deletion of the DEFB126 (rs11467417) in Iranian infertile males with a recurrent history of CT. Methods: Semen samples of 1080 subfertile males were investigated. Among patients who had CT-positive results, sperm DNA from 50 symptomatic and 50 asymptomatic patients were collected for the DEFB126 genotype analysis. Additionally, a control group comprising 100 DNA samples from individuals with normal spermogram and testing negative for CT was included in the study. The PCR-sequencing technique for detecting the 2-nt deletion of the second exon of the DEFB126 was performed. Results: The Chi-squared test comparing all three groups revealed no significant difference across the different genotypes. Moreover, no significant difference between the symptomatic and asymptomatic groups was seen. However, analysis within CT-positive patients and controls demonstrated significant difference between the frequencies of homozygous del/del. Conclusion: The higher frequency of the 2-nt deletion of the DEFB126 in CT-positive patients suggests that the occurrence of mutations in the DEFB-126 may cause the impairment of the antimicrobial activity of the DEFB126 protein and consequently makes individuals more susceptible to infections such as CT.