Streptococcus agalactiae is a significant co-pathogenic bacterium in humans and animals, including fish. Bacteria secrete a variety of proteins through an accessory secretion system to modulate their interactions with the host. To investigate the role of the accessory secretion system in S. agalactiae, a deletion mutant strain (ΔaccSec) was constructed via homologous recombination. The accessory secretion system was found to be essential for the viability of S. agalactiae, and its absence led to increased cell death and lysis. In the extracellular fraction of the ΔaccSec mutant, a reduction in the secretion of 33 proteins was observed. Analyses of biological properties indicated that ΔaccSec exhibited significantly reduced stress tolerance and envelope stability. Pathogenicity experiments demonstrated that the ΔaccSec mutant had significantly lower adhesion to cells and fish tissues, as well as decreased resistance to whole blood killing and phagocytosis by macrophages. The cumulative mortality of ΔaccSec in tilapia after intraperitoneal injection was reduced by 55.3-74.2 %. The ΔaccSec mutant exhibited a markedly diminished capacity for colonization in tilapia. Furthermore, we found that ΔaccSec mutant induced higher macrophage reactive oxygen species (ROS) levels and significantly upregulated MHC-II, TLR-2 transcript levels in tilapia spleens compared to the wild-type. Overall, these findings underscore the importance of the accessory secretion system in S. agalactiae pathogenicity, particularly in stabilizing the bacterial envelope, facilitating adhesion, and evading host immunity. The results of this study provide new insights into the mechanisms of virulence regulation in S. agalactiae and lay a foundation for developing live attenuated vaccines.