Masculinized females, named sex-reversed females (SRF), have a male phenotype but retain the female genotype (XX) and all spermatozoa produced in their testes carry the X chromosome. Masculinization of females leads to incomplete testicular development and the production of lower-quality semen. The mechanism of masculinization is unknown at present. Therefore, the aim of our study was to identify differentially abundant proteins in testicular semen of normal males and SRF using a difference in-gel electrophoresis approach. Masculinization seemed to not lead to significant changes in the testicular seminal plasma proteome, but did have an impact on the proteome of SRF and normal male sperm. We identified 26 proteins enriched ( < 0.05) in testicular male spermatozoa compared to SRF. A total of 28 proteins were also found to be differentially expressed ( < 0.05) in testicular SRF sperm in comparison to normal males. Bioinformatic analysis highlighted pathways associated with energy production for normal male spermatozoa and pathways related to protein remodeling for SRF sperm. Normal male spermatozoa seemed to be equipped with proteins participating in diverse metabolic pathways, focusing on producing the energy required for sperm movement. On the other hand, SRF spermatozoa were characterized by the enhanced expression of proteins associated with cytoskeletal structures and those related to remodeling, which could indicate that spermatogenesis and spermiogenesis are not fully accomplished. These results can be the basis for further research on the molecular mechanisms of masculinization and toward the development of a method for separating X and Y fish sperm.