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An increasing body of data implicates the Septin family in the pathology of several diseases, including male fertility. The objective of this study was to evaluate the gene and protein expression pattern of Septin 14 in the testis tissue of azoospermic men. In addition, Septin 14 localization was also assessed in the sperm. Testicular tissues were obtained from biopsies of non-obstrutive azoospermic men who underwent diagnostic testicular biopsy in Royan institute and were divided into two groups: TESE + with positive result in testicular sperm extraction (with hypospermatogenesis pathology) and TESE- with negative result (included patients with Sertoli cell only syndrome and maturation arrest pathologies). Total RNA and protein was extracted using trizol reagent. Septin 14 gene and protein expression level were assessed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot techniques, respectively. The localization of Septin 14 protein was also studied by Immunocytochemistry. The expression of Septin 14 was significantly lower (p < 0. 05) in TESE- group than TESE + in both mRNA and protein levels. The localization of Septin 14 protein was detected in the head to tail of normal sperms with high localization in front of the acrosome and the neck. This is a novel localization report on Septin 14 in sperm. Regarding the presence of this protein in the sperm acrosome and neck, it can be concluded that decreasing of Septin 14 protein expression may be associated with the pathogenesis of male infertility and therefore Septin 14 expression level maybe critical for human spermatogenesis., Competing Interests: Declaration of Competing Interest There is no Conflict of Interest., (Copyright © 2020. Published by Elsevier B.V.)