Aromatase is the rate limiting enzyme in estrogen biosynthesis encoded by the CYP19A1 gene, and excessive aromatase can reduce the semen quality of roosters. Seminal plasma extracellular vesicles are tiny membrane vesicles that can carry and transmit signaling molecules, thereby affecting semen quality. It is still unclear whether aromatase affects the semen quality through SPEV. In this study, we injected lentivirus carrying CYP19A1 gene over-expression vector into the testis of 35 -week-old roosters and measured semen quality, serum and seminal plasma T and E2 levels. Subsequently, we isolated seminal plasma extracellular vesicles and performed proteomic sequencing, aiming to reveal the molecular mechanisms of aromatase mediated seminal plasma extracellular vesicles regulation of rooster semen quality.