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Objective In this study, we evaluated the effects of promoter methylation of MTHFR on oligozoospermia risk, followed by an in silico analysis. Materials and Methods In a case-control study, semen samples were collected from infertile and healthy control men. MTHFR promoter region was amplified by methylation-specific polymerase chain reaction (PCR). Finally, the promoter region of MTHFR was analyzed by bioinformatics software. Results Our data revealed significant associations of CpG island promoter methylation with oligozoospermia in a case-control study.