Interleukin-15 (IL-15) is a potent growth factor for activated T and natural killer (NK) cells, stimulator of memory T cells and plays an important role in viral immunity. To investigate mechanisms underlying the antiviral activity of IL-15, a recombinant vaccinia virus (rVV) encoding murine IL-15 (VV-IL-15) was constructed. Following infection of mice with VV-IL-15, virus titres in the ovaries were significantly reduced compared to mice infected with control VV. Growth of VV-IL-15 was also reduced in nude athymic mice, indicating the antiviral activity of IL-15 does not require T cells. Additionally, VV-IL-15 augmented the cytolytic activity of natural NK cells in the spleen and enhanced interferon (IFN) mRNA expression and transcription factors associated with IFN induction. Using knockout mice and antibody depletion studies, we showed for the first time that the control of VV-IL-15 replication in mice is dependent on NK cells and IFNs and, in their absence, the protective role of IL-15 is abolished.