We have developed highly potent synthetic activators of the vertebrate immune system that specifically target the RIG-I receptor. When introduced into mice, a family of short, triphosphorylated stem-loop RNAs (SLRs) induces a potent interferon response and the activation of specific genes essential for antiviral defense. Using RNA sequencing, we provide the first in vivo genome-wide view of the expression networks that are initiated upon RIG-I activation. We observe that SLRs specifically induce type I interferons, subsets of interferon-stimulated genes (ISGs), and cellular remodeling factors. By contrast, polyinosinic:polycytidylic acid [poly(I:C)], which binds and activates multiple RNA sensors, induces type III interferons and several unique ISGs. The short length (10 to 14 base pairs) and robust function of SLRs in mice demonstrate that RIG-I forms active signaling complexes without oligomerizing on RNA. These findings demonstrate that SLRs are potent therapeutic and investigative tools for targeted modulation of the innate immune system.