Abstract: Viral nervous necrosis (VNN) caused by a betanodavirus (NNV) is one of the major diseases in Asian seabass (Lates calcarifer) hatcheries. Our previous studies showed that the tbx21 gene was in a QTL for NNV resistance in linkage group 23 in Asian seabass. The expression of this gene was changed in tissues of Asian seabass challenged with NNV. However, the role of tbx21 in NNV resistance remains largely unknown. In this study, tbx21 of Asian seabass was characterized. This gene consists of an ORF of 1866 bp, a 5′ UTR of 357 bp, and a 3′ UTR of 4674 bp. The TBX21 protein showed substantial amino acid similarity (70–96%) with other fish but exhibited lower identity (47–52%) with mammals. One SNP identified in the first intron was significantly associated with NNV resistance. In healthy fish, tbx21 was expressed in all tissues examined, and was highly expressed in the kidney and liver. The expression of tbx21 increased in the eye, gills, heart, kidney and gut, but decreased in the brain and spleen at five days after NNV challenge. Overexpression of tbx21 reduced the replication of NNV, whereas knockdown increased viral expression and virus titers. These results suggest that tbx21 plays a key role in NNV resistance. The SNP in this gene could be used as a marker to facilitate marker-assisted selection for NNV resistance. Further investigation of polymorphisms in the 5' and 3' UTRs of tbx21 may provide additional insights into the gene's role in NNV resistance.