Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT protein family that controls cell proliferation, differentiation and inflammation. Recent studies have found that STAT3 also plays a key role in regulating host antiviral immune response. In order to study the roles of STAT3 in teleost antiviral response, two cDNA sequences encoding STAT3 (AjSTAT3-L and AjSTAT3-S) were obtained from Anguilla japonica, by reverse transcription polymerase chain reactions (RT-PCR) and rapid amplification of cDNA ends PCR (RACE-PCR) experiments. The open reading frames of AjSTAT3-L and AjSTAT3-S are 2349 bp and 1470 bp in length, coding 782 and 489 amino acids (aa), respectively. Gene structure analysis revealed that AjSTAT3-L consists of 23 exons and 22 introns, while AjSTAT3-S was generated by alternative splicing lacking the exons 2-7 and exon 21. Similar to its mammalian counterpart, the predicted protein sequence of AjSTAT3-L possesses an N terminal domain (NTD, 1-120 aa), a coiled coil domain (CCD, 104-313 aa), a DNA binding domain (DBD, 325-462 aa) and a Src homology 2 domain (SH2, 577-672 aa). Whereas, AjSTAT3-S lacks the entire CCD and C-terminal tail sequence of NTD (69 aa). Phylogenetic analysis showed that AjSTAT3-L and AjSTAT3-S formed a basal clade to teleost STAT3, and vertebrate STAT3 genes were grouped together to form a clade that was sister to the clade of STAT1 and STAT4. Subcellular localization experiments showed that AjSTAT3-L and AjSTAT3-S are predominantly localized in cytoplasm. Luciferase reporter assays were performed to study the effect of AjSTAT3-L and AjSTAT3-S on the transcriptional activity of antiviral genes, showing that the overexpression of AjSTAT3-L or AjSTAT3-S suppressed the Poly I∶C-induced activation of IFN and Mx promoters. Taken together, we have identified AjSTAT3 and its transcript variant, a novel isoform of STAT3 generated by alternative splicing. Both isoforms are functionally relevant and played a negative regulatory role in fish antiviral immune response.