In some vertebrate species, type I interferon(IFN)-induced Mx gene expression has been shown to confer resistance to some single-stranded RNA (ssRNA) viruses in vitro. Because the bovine species is subject to an exceptionally wide array of infections caused by such viruses, it is anticipated that an antiviral allele should have been retained by evolution at the bovine Mx locus. The identification of such allele may help in evaluating the real significance of the Mx genotype for disease resistance in vivo, in deciphering host-virus molecular interactions involved, or in improving innate disease resistance of livestock through marker-assisted selection. We validated a double transgenic Vero cell clone in which the bovine Mx1 reference allele is placed under control of the human cytomegalovirus (CMV) enhancer-promoter sequence containing elements from the bacterial tetracycline resistance operon to regulate transcription. In the selected clone, transgene repression was very tight, and derepression by doxycycline led to homogeneous 48-h duration expression of physiologic levels of bovine Mx1. Expression of the transgene caused a dramatic decrease in cytopathic efficiency and a 500-5000-fold yield reduction of the Indiana and New Jersey serotypes of vesicular stomatitis virus (VSV). To our knowledge, the transgenic clone developed here is the first ever reported that allows conditional expression of an Mx protein, thus providing a valuable tool for studying functions of Mx proteins in general and that of bovine Mx1 in particular. This latter may henceforward be included in the group of Mx proteins with authenticated anti-VSV activity, which offers new research avenues into the field of host-virus interactions.