Sperm storage in females is widespread among vertebrates and insects, and the expression of proteins in the female reproductive tract is influenced by the presence of sperm, allowing for adaptation to this phenomenon. Through histological observation, we confirmed that sperm were stored in the isthmic fossa outside the oocyte during the post-mating (POM) stage, and closer to the epithelial cells during the pre-fertilization (PRF) stage. In addition, we observed asynchronous ovarian development in black rockfish, where oocytes at various stages could be identified during the PRF phase. This study investigated the ovarian protein expression changes in black rockfish (Sebastes schlegelii) during key reproductive stages: pre-mating (PRM), POM, unmated control (POM-CT), and PRF. A total of 5012 proteins were identified, with notable fluctuations in protein expression observed at the PRF stage. Specifically, 140 proteins were upregulated and 615 downregulated when compared to the PRM stage, while 101 proteins were upregulated and 531 downregulated in comparison to the POM stage. The functional enrichment analysis of differentially expressed proteins (DEPs) revealed distinct pathways: POM vs. PRM showed involvement in vesicle sorting and hormone signaling; PRF vs. POM indicated pathways related to chromatin remodeling and gene expression regulation; and POM vs. POM-CT highlighted pathways associated with immune response. These findings suggested that these signaling pathways may play a crucial role in oocyte development and sperm storage. The majority of DEPs were localized in the nucleus, with key interactions involving proteins such as GSK3B and MED1. These findings enhance our understanding of the molecular mechanisms underlying oocyte maturation and sperm storage, providing insights relevant to reproductive biology and aquaculture practices.