Semen analysis is used for diagnosing male infertility and evaluating male fertility for more than a century. However, the semen analysis simply represents the population characteristics of sperm. It is not a comprehensive assessment of the male reproductive potential. In this study, 20 semen samples from human sperm bank with distinctive artificial insemination with donor sperm (AID) clinical outcomes were collected and analyzed using a two-dimensional differential in-gel electrophoresis (2D-DIGE); 45 differentially expressed protein spots were obtained, and 26 proteins were identified. Most differentially expressed proteins were related to sperm motility, energy consumption, and structure. These identified proteins included several sperm proteins associated with the nucleus on the X chromosome (SPANX) proteins. This prospective study aimed to investigate the association between the expression levels of SPANX proteins and the AID clinical outcomes. The proteins identified in this study provided a reference for the molecular mechanism of sperm fertility and revealed a predictive value of the SPANX proteins.