Background: Infertility is the inability of a couple to conceive after one and a half years of unprotected sex. Male infertility, which accounts for almost half of infertility cases, is considered as a major problem all over the world. The aim of this study was to investigate the association of CYP1A1 polymorphisms with idiopathic non-obstructive azoospermia in a South Indian cohort. Materials and Methods: An experimental study was conducted with idiopathic nonobstructive azoospermia. A total of 120 infertile and 80 fertile samples were collected, and DNA was then extracted from all samples. The CYP1A1 *2.4 polymorphism genotyp- ing was carried out by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Results: The genotype distribution of CYP1A1 *2A polymorphism showed significant difference between patients and controls. Moreover, the CC genotype was associated with decreased risk of idiopathic non-obstructive azoospermia in comparison with the TT and TC genotypes. Conclusion: The current experimental study identified that the CT genotype of CYP1A1*2A polymorphism may contribute to the pathogenesis of male infertility in the South Indian population. © 2017, Royan Institute (ACECR). All rights reserved.