Polio or poliomyelitis is a disabling and life-threatening disease caused by poliovirus (PV). As a consequence of global polio vaccination efforts, wild PV serotypes 2 and 3 have been eradicated around the world, and wild PV serotype 1-transmitted cases have been largely eliminated except for limited regions. However, vaccine-derived PV, pathogenically reverted live PV vaccine strains, has become a serious issue. For the global eradication of polio, the World Health Organization is conducting the third edition of the Global Action Plan, which is requesting stringent control of potentially PV-infected materials. To facilitate the mission, we generated a PV-nonsusceptible Vero cell subline, which may serve as an ideal replacement of standard Vero cells to isolate emerging/re-emerging viruses without the risk of generating PV-infected materials.