Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fishes, which is poorly understood, largely because its causative iridovirus, erythrocytic necrosis virus (ENV), is intractable to cell culture. Natural VEN epizootics and observations studies in wild populations suggest that temperature may be an important disease cofactor. Here, a controlled laboratory exposure study provides evidence for a direct relationship between temperature and the progression of viral erythrocytic necrosis (VEN) in Pacific herring. Waterborne exposure of Pacific herring to kidney homogenates containing ENV resulted in the establishment of infections, characterized by high infection prevalence (89%; 40/45) and mean viral loads (5.5 log10- gene copies / ug DNA) in kidney tissues at 44 d post exposure. Viral loads were higher in herring from the ambient (9.0 °C) and warm (13.5 °C) treatments (6.1 - 6.2 log10- gene copies / total DNA) than from the cool (6.9 °C) treatment (4.3 log10- gene copies / total DNA). Similarly, the peak proportion of diseased fish was directly related to temperature (P 0.001), with cytoplasmic inclusion bodies detected in 21% of herring from the cool, 52% from the ambient, and 60% from the warm treatments. The mean disease load in each fish (enumerated as the percent of erythrocytes with cytoplasmic inclusions), increased with temperature from 15% in the cool, 36% in the ambient, and 32% in the warm treatments at 44 days post exposure. Transcriptional analysis indicated that the number of differentially expressed genes among ENV-exposed herring increased with temperature, time post exposure, and viral load. Correlation network analysis of transcriptomic data showed robust activation of interferon and viral immune responses in hepatic tissue of infected individuals independent of other experimental variables.