Rainbow trout (Oncorhynchus mykiss) is an important cold-water fish widely cultivated in China. The frequent occurrence of viral diseases caused by infectious hematopoietic necrosis virus (IHNV) seriously restricted the healthy development of the rainbow trout farming industry. However, the immune defense mechanism induced by IHNV in rainbow trout has not been fully elucidated. In the present study, we detected mRNA and miRNA expression profiles in rainbow trout head kidney after IHNV infection and the expression patterns of key immune-related genes at different post-infection periods (0-, 6-, 12-, 24-, 48-, 72-, 96-, 120- and 144 hours post-infection (hpi)) using RNA-seq and qRT-PCR. The results showed that a total of 7486 genes and 277 miRNAs were differentially expressed, and numerous differentially expressed genes (DEGs) enriched in the immune-related pathways such as Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction and JAK-STAT signaling pathway were significantly up-regulated, including LGP2, MDA5, TRIM25, IRF3, IRF7, TLR3, TLR7, TLR8, MYD88, IFN1, MX1 and VIG-1. Integration analysis identified six miRNAs (miR-141-y, miR-200-y, miR-144-y, miR-2188-y, miR-725-y and miR-203-y) that target at least four key immune-related genes (TRIM25, LGP2, TLR3, TLR7, IRF3 and IRF7). Expression analysis showed that TRIM25, MDA5, LGP2, TLR3, TLR7, IRF3, IRF7, IFN1, MX1 and VIG-1 genes increased firstly and then decreased, and reached peak expression at 48 hpi. These findings improve our understanding of the immune mechanism of rainbow trout infected with IHNV, and provide a basic data for future breeding for disease resistance in rainbow trout. Overall design: In the present study, we detected mRNA and miRNA expression profiles in rainbow trout head kidney after IHNV infection and the expression patterns of key immune-related genes at different post-infection periods (0-, 6-, 12-, 24-, 48-, 72-, 96-, 120- and 144 hours post-infection (hpi)) using RNA-seq and qRT-PCR.