Streptococcus agalactiae (group B streptococcus, GBS), a broad-spectrum pathogen, causes great economic losses in fish aquaculture, especially the industry of tilapia. Until now, the knowledge of the immune response mechanism against S. agalactiae infection in tilapia has been limited. In the present study, the gill transcriptome of the tilapia from the GBS and the phosphate buffered saline (PBS) groups were sequenced. The transcriptomic analysis results presented the differentially expressed genes (DEGs) at different time points (DEGs number, 6 h: 2122, 9 h: 1851, 15 h: 1791, and 18 h: 2395) after GBS injection, and significantly enriched immune-related gene ontology (GO) terms such as the innate immune response. The significantly enriched immune pathways included the Toll-like receptor signaling pathway, the nucleotide oligomerization domain (NOD)-like receptor signaling pathway, the cytosolic-DNA sensing pathway, and the intestinal immune network for Immunoglobulin A (IgA) production. Most of the DEGs in Toll-like receptor signaling, NOD-like receptor signaling, and cytosolic-DNA sensing pathways presented upregulations at 18 h, which indicated that the innate immune pathways were activated. Two immune-related pathways (phagosome and cell adhesion molecules) were significantly enriched at all time points, suggesting that these two pathways might also play important roles in the immune response against the GBS infection. The results of HE staining showed that the gills of tilapia were damaged seriously at 9 h post-infection, which might be due to the possibility of pyroptosis resulting from the changes of DEGs in the NOD-like receptor signaling pathway. This study provided new insight into the mechanisms of gill damage in fish infected with S. agalactiae.