Interferon regulatory factor 3 (IRF3) is an important member of the interferon regulatory factor family and plays an important role in defensing against pathogen infection. To investigate the function of the IRF3 gene in Apostichopus japonicus, we obtained the full-length cDNA sequence of the IRF3 gene in A. japonicus by cloning and sequence splicing using RACE technology, and named it AjIRF3. The full-length sequence of AjIRF3 gene is 2 023 bp and encodes 441 amino acids. Sequence analysis revealed that the C terminus of AjIRF3 contains an IAD domain. Phylogenetic tree analysis indicated that AjIRF3 is a new member of the IRF3 family and is more closely related to the invertebrate mollusc Littorina littorea. Tissue distribution analysis showed that AjIRF3 is expressed in all tissues, with the highest expression in muscle, followed by intestine, and the lowest expression in coelomocyte. Furthermore, the expression of AjIRF3 in coelomocyte was significantly up-regulated under Vibrio splendidus infection and reached its highest level at 6 h, which was 3.64-fold higher (P<0.05) than that of the control. Moreover, AjIRF3 mRNA expression levels peaked at 48 h after lipopolysaccharide (LPS) and poly(I:C) infection, increasing by 3.20-fold (P<0.01) and 8.57-fold (P<0.01), respectively, compared with the control. Subcellular localization studies showed that AjIRF3 is distributed in the cytoplasm, but it is mainly translocated from the cytoplasm to nucleus after V. splendidus infection. Additionally, we found that the apoptosis rate of coelomocyte was significantly decreased by 16% (P<0.01) following AjIRF3 interference. More importantly, our result showed that the number of intracellular bacteria in the si-AjIRF3 group was significantly increased by 19.83-fold (P<0.05) compared with NC group after V. splendidus infection. Collectively, these findings suggest that AjIRF3 plays important roles in innate immunity and regulates apoptosis to resist pathogen infection.