Disease caused by grouper iridovirus (GIV) has resulted in economic losses due to high mortality in grouper culture. Thirty-eight up- and 48 down-regulated known entities have been identified using a GIV-infected grouper kidney cDNA microarray chip. Further quantitative validation was executed in the head-kidney and spleen for 24 candidate genes and 7 immune factors following GIV inoculation. Significant induction with various patterns could be seen in 30 tested genes in the spleen. However, only 23 genes had induction in the head-kidney and meanwhile 5 genes showed reduction. Transcriptional expression profiles of selected genes in response to lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (PIC) were also established to compare with the GIV-stimulated expression. The results indicated that the responses of most genes facing GIV invasion have more similarities to PIC treatment than LPS. Seven genes are thought to be interferon-related factors: RNA helicase DHX58, ISG15, viperin, HECT E3 ligase (HECT), CD9, urokinase plasminogen activator surface receptor (PLAUR) and Mx-1. Following immunization with inactivated GIV, significant induction could be seen in DHX58, viperin, IL-1β, IL-8, COX-2, HECT, PLAUR, IgM, Mx-1, very large inducible GTPase-1 (VLIG1) and TNF-α in the head-kidney or spleen, and the latter 6 genes also had a gradual increasing pattern by a boosting immunization. These factors might play important roles in adaptive antiviral protection. Thus, we have characterized the temporal response patterns of virus responsive genes and have also identified several potential immune markers to further investigate host antiviral defense mechanisms.