Aim: The potential mechanisms underlying unexplained infertility, which is a condition in which couples experience infertility without an identifiable cause in both men and women, have not yet been clarified. The present study aimed to investigate the role of Sirtuin 1 (SIRT1) gene in male infertility and unexplained infertility. Methods: A total of 50 couples who applied for infertility evaluation and treatment were included. Medical histories were obtained, and fertility status was assessed. Semen analysis results, evaluated according to World Health Organization (WHO, 2010) criteria, were categorized into low and normal semen parameter groups based on sperm count and motility. Couples were further classified into three groups based on fertility status: Non-infertility group (n = 13), comprising couples without clinical infertility. Male infertility group (n = 19), comprising couples with infertility attributed to a male factor. Unexplained infertility group (n = 18), comprising couples with infertility for which no identifiable cause could be determined in either partner. Changes in SIRT1 gene expression among the groups were analyzed using qPCR. Results: SIRT1 gene expression was not associated with sperm count or motility parameters. However, expression of SIRT1 was significantly reduced in the male infertility group and, more notably, in the unexplained infertility group compared to the non-infertility group. Conclusions: Our study is the first to demonstrate that decreased SIRT1 expression in human mature spermatozoa may play a significant role in fertilization potential by influencing functions of sperm other than count and motility, highlighting a novel potential mechanism underlying unexplained infertility.