Despite the promise of small interfering RNAs (siRNAs) in antiviral therapy, few in vivostudies of them as inhibitors of viral replication and disease have been published, a lack that is most probably due to problems with obtaining successful delivery. Here we introduce a novel in vivomodel composed of small juvenile rainbow trout and a fish pathogenic virus to analyze the delivery and antiviral effects of formulated siRNAs. Intraperitoneally (IP) injected siRNAs formulated in polycationic liposomes, and to a lesser degree naked siRNAs, primarily entered free IP cells, including macrophage-like cells. Uptake in these cells correlated with antiviral activity, seen as reduced mortality of virus-challenged fish. However, protection at the disease level was not dependent upon which of three tested siRNAs was used, and protection correlated with up-regulation of an interferon (IFN)-related gene in the liver, indicating a systemic IFN response. The results emphasize the compromise in using transfection reagents for improved uptake of siRNAs, where these reagents also increase the risk of the siRNAs ending up in a cellular compartment in which stimulation of non-specific anti-viral defence mechanisms will be initiated.