Abstract: 【Objective】 This study aimed to investigate the structural characteristics and tissue expression pattern of the interferon a3 （IFNa3） gene in GIFT Oreochromis niloticus， to examine its expression change after polyinosinic acid Poly（I：C） stimulation and tilapia lake virus （TiLV） infection， and to discover effects of IFNa3 gene on interferon-stimulated genes （ISGs） and TiLV replication， providing a theoretical basis for disease prevention and control strategies in tilapia aquaculture.【Method】 The IFNa3 gene in GIFT Oreochromis niloticus was cloned and subjected to bioinformatic analysis， and real-time fluorescence quantitative PCR was employed to examine expression distribution of IFNa3 gene in healthy tissues of GIFT Oreochromis niloticus and expression change after Poly（I：C） stimulation and TiLV infection. Effects of IFNa3 of GIFT Oreochromis niloticus on ISG expression and TiLV replication were evaluated by gene experiments of overexpression and knockdown.【Result】 The open reading frame （ORF） of IFNa3 gene of GIFT Oreochromis niloticus was 570 bp， encoding 189 amino acid residues. The IFNa3 protein contained one IFabd domain， showing typical structure characteristic of typeⅠIFN. The similarity between IFNa3 amino acid sequence of GIFT Oreochromis niloticus and typeⅠIFNa3 amino acid sequence in other teleost fish species was 70.3%-83.5%， with the highest similarity observed with the IFNa3 amino acid sequence of Haplochromis burtoni （83.5%）. The IFNa3 gene was expressed in tissues of spleen， liver， brain， gill， intestine， muscle， heart， and head kidney， with the highest expression in gill tissues. After Poly（I：C） stimulation and TiLV infection， IFNa3 expression was up-regulated in the gill， head kidney， and spleen， displaying a temporal pattern of initial increasing and subsequent decreasing. The overexpressed gene IFNa3 of tilapia brain （TiB） cells could extremely up-regulate the expression of STAT1， ISG15， and Mx （P<0.01， the same below）， while extremely down-regulate the relative expression of TiLV-S10 gene. After knocking down the expression of IFNa3 gene by siRNA， the relative expression of TiLV-S10 was extremely significantly increased， but the expressions of STAT1， ISG15， and Mx genes were extremely significantly decreased.【Conclusion】 The IFNa3 of GIFT Oreochromis niloticus exhibits typical structure characteristics of typeⅠIFN， and is up-regulated in tissues of gill， head kidney， and spleen after immune stimulation. It activates JAK-STAT signaling pathway， and induces multiple downstream expressions of ISGs， and suppresses TiLV replication at the cellular level， thereby playing a critical role in the innate immune defense against TiLV infection in GIFT Oreochromis niloticus.