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Male infertility evaluation is mainly based on semen analysis. Thus, identification of additional diagnostic methods is valuable. The aim of this study was to analyse the sperm proteome of infertile men to identify the underlying mechanisms and reliable diagnostic biomarkers. This cross‐sectional study consisted of 16 infertile men and seven proven fertile men. An LC‐MS/MS approach was performed in five pooled samples of each group (proven fertile men, primary infertility and secondary infertility). Differentially expressed proteins were used for functional enrichment analyses, and the most central proteins involved in altered functions in both infertile groups and the testis‐specific proteins were validated using Western blotting and immunocytochemistry. In total, 1,305 sperm proteins were identified, of which 102 were underexpressed and 15 were overexpressed proteins in both infertile groups. Underexpressed …