Abstract: 【Objective】This study aimed to clone the claudin-3 gene of Oreochromis niloticus， construct an in vitro blood-brain barrier （BBB） model， and analyze the expression changes of claudin-3 gene after Streptococcus agalactiae infection， with an aim to provide theoretical references for investigating the role of claudin-3 gene in the process of Oreochromis niloticus resisting Streptococcus agalactiae infection.【Method】The claudin-3 gene was cloned， followed by sequence analysis and bioinformatics analysis. Tissues including skin， muscle， liver， spleen， head kidney， gill， brain， and intestine of Oreochromis niloticus infected with ZQ0910 were collected to detect the tissue expression profile of claudin-3 gene by real-time fluorescence quantitative PCR. An in vitro BBB model was constructed and infected with ZQ0910 and SA068， and changes of relative expressions of claudin-3 gene were detected by real-time fluorescence quantitative PCR.【Result】The claudin-3 gene of Oreochromis niloticus was cloned， with a full-length of 648 bp， encoding 216 amino acid residues. The claudin-3 protein contained 17 amino acid functional sites and four transmembrane domains. The claudin-3 protein of Oreochromis niloticus had the highest homology with that of Oreochromis aureus， with a similarity of 99.53%； phylogenetic tree analysis showed that it clustered into a large clade with claudin-3 proteins from multiple species， including mammals. The claudin-3 gene had relatively high expressions in the liver， gill， head kidney， brain， and muscle tissues of Oreochromis niloticus， and low expressions in the skin， spleen， and hindgut tissues. At 3 h after ZQ0910 infection， the relative expression of claudin-3 gene increased significantly in skin， liver， spleen， and hindgut tissues （P<0.05， the same below）， while it significantly decreased and recovered after 12 or 24 h in brain， muscle， and head kidney. At 12 h after ZQ0910 infection， the relative expression of claudin-3 gene in the TA-02 monoculture group was significantly higher than that in the BBB model group. The relative expression of claudin-3 gene in the BBB model group remained stable during 0-12 h after SA068 infection， and a significant increase began after 24 h.【Conclusion】The claudin-3 gene is a key factor for Oreochromis niloticus to resist the invasion of Streptococcus agalactiae into the blood-brain barrier， with tissue-specific expression. The gene exhibits dynamic regulation after Streptococcus agalactiae infection， especially affected by astrocytes in the in vitro BBB model. The claudin-3 gene may participate in the immune defense of the central nervous system by regulating the integrity of tight junctions， and the changes in its relative expression may reflect the interaction between the adaptive regulation of host barrier function and pathogen invasion.