Objective：To establish and optimize a set of two-dimensional gel electrophoresis（2-DE） techniques for proteomic research of sperm protein of varicocele patients. Methods：Spermatozoa in the semen were purified by Percoll gradient technique. The effects of different sample preparation methods，different gels，different amount of protein loads and different rehydration methods to the 2-DE maps were compared. Results：After the sample was treated with clean-up kit，a high resolution and clear 2-DE map for sperm pro－tein of varicocele patients was obtained with pH 5~8 gel，250 μg protein loads and initiative rehydration method. Conclusion：The 2-DE map of sperm protein of varicocele patients is successfully obtained with good repeatability and high resolution，which paves the way for the further proteomic research of infertility caused by varicocele.