Nile tilapia is an important economically farmed fish worldwide, and Streptococcus agalactiae has become the most serious disease problem for the tilapia industry in China. Therefore, a better understanding of the innate immune mechanisms and their functions in the context of pathogen invasion is needed. In this study, a high-throughput proteomic strategy was developed for determining the differential expression proteins (DEPs) of the genetically improved farmed tilapia (GIFT) (susceptible and resistant GIFT species) splenic responses to S. agalactiae infection. The results showed 1608 proteins were identified with high confidence (P < 0.05). A total of 441 and 382 DEPs were characterized in susceptible and resistant GIFT tilapia species, respectively. Gene ontology (GO) annotation showed that the DEPs covered many biological functions, including cellular process, metabolism, and immune process. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the DEPs were mainly enriched in the lysosome, apoptosis, and complement and coagulation cascade pathways during S. agalactiae infection. By group comparisons, the proteins related to immune response, cytoskeleton, and energy metabolism of disease-resistant strains were upregulated compared with those of susceptible strain. The content determination of immune-related enzyme activity was consistent with the results obtained throughout the study. More active innate immune system and cell defense is the basis of S. agalactiae resistance for disease-resistant GIFT tilapia species. Furthermore, 34 proteins involved in disease resistance were characterized. Therefore, the present study contributes to a better understanding of the immune system and defense mechanisms of GIFT tilapia in response to S. agalactiae infection and provides valuable recommendations for breeding disease-resistant GIFT tilapia species.