Objective: To compare the sperm protein profile between men with and without varicocele. Methods: The present study was designed as a case-control study. The research patients were recruited from the Infertility Unit of the Royan Institute in 2009. We included 20 sperm samples from normozoospermic men without varicocele (control group) and 20 sperm samples from oligozoospermic patients with varicocele, grade 3 (varicocele group) in the present study. The sperm protein profile in the 2 groups was characterized using 2-dimensional gel electrophoresis. Differences in protein expression were established using gel analysis software, and protein identification was performed using mass spectroscopy analysis. Results: In the varicocele group, we noted 15 consistent differences in protein expression (1, spots missing; 12, less abundant; and 2, more abundant) compared with the control group (P <.01). The findings revealed that heat shock proteins, mitochondrial proteins, and cytoskeleton proteins are the proteins mainly affected by varicocele disease. Conclusion: To our knowledge, the present study is a novel study, with few studies describing the correlation between sperm protein in men with and without varicocele obtained using a 2-dimensional proteomic approach. It could be an important prerequisite to the development of diagnostic tests to predict varicocelectomy outcomes in patients with varicocele and abnormal findings on a spermogram in the clinical environment.