Equine interferon-gamma (eIFN-g) expressed both in E. coli and baculovirus were evaluated for antiviral activity against recombinant Vesicular Stomatits Virus expressing green fluorescence protein (rVSV-GFP) in EFK-78 cells. The assays were conducted in 96-well plate. Virus infectivity was measured by quantifying GFP-positive cells, instead of quantifying the CPE reduction. Prior to infection of EFK-78 cells with rVSV-GFP, the cells were incubated with eIFN-g. The GFP expression in the EFK-78 cells dramatically decreased in the cells treated with eIFN-g in a dose-dependent manner, comparing with the mock-treated cells. The titers of antiviral activity were 1×103AU/mL and 1×105AU/mL of eIFN-g expressed from E. coli and baculovirus, respectively. The antiviral activities of the recombinant eIFN-g were highly efficient and specific, as it was blocked by mAbs against eIFN-g.