In order to understand the structure and expression profiles of TNFSF6 gene in Gadus macrocephalus, the full-length cDNA of TNFSF6 was cloned and analyzed using the bioinformatics tools, and quantitative real-time PCR (qRT-PCR) was used to examine the TNFSF6 mRNA expression levels in various tissues of adult G. macrocephalus, at different development stages of larvae, and during PCNNV outbreak. The results showed that TNFSF6 cDNA contains a 5'UTR of 315 bp, a 3'UTR of 500 bp, and an ORF of 573 bp encoding a protein of 190 amino acids. The results revealed that TNFSF6 was expressed in various tissues and it was higher in the spleen and gill. QRT-PCR indicated that the transcript level of TNFSF6 on 15, 20, 37, and 40 d post hatching was 0.28, 0.15, 0.12, 0.13 times of that on 5 d respectively. Compared to the control, the transcript level of TNFSF6 decreased significantly during virus outbreak on 77 d, however, there was an upward trend of TNFSF6 expression on 24 and 46 d respectively. The results indicated that TNFSF6 plays an important role in the early development stages and in responding to PCNNV outbreak.