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Purpose: The Importance and key role of Wnt/β-catenin signaling pathway in spermatogenesis is known. Abnormalities of this pathway in Sertoli and germ cells leads to infertility. Leydig cells play an important role in spermatogenesis and male reproduction. As of now, exact position of the Wnt/β-catenin signaling pathway disorders in the tissue and possible involvement of Leydig cells has not been investigated. Methods: Samples of our previous study were used for common Y chromosome microdeletions screening and common CFTR gene mutations.1 β-catenin gene expression were evaluated and compared between testicular tissue obtained by testicular sperm extraction (TESE) in two groups of obstructive (n=10) and non-obstructive (n=10) azoospermic infertile men. Location of β-catenin accumulation was detected by immunofluorescence technic and quantitatively compared in the tissue followed by counterstaining with anti-vimentin antibody. It was used as specific marker of leydig cells to determine and confirm the cells in which this gathering was occurred. Results: β-catenin gene expression does not have a significant difference between the obstructive azoospermia (0.998) and non-obstructive azoospermia group (0.891). β-catenin was abnormally aggregated in leydig cell of non-obstructive azoospermic men. Conclusion: Gathering β-catenin in cytoplasm of leydig cells can disrupt spermatogenesis and cause infertility in men.