Saffold viruses (SAFV) have been discovered recently and they are classified into Theilovirus species in genus Cardiovirus in the Picornaviridae family. SAFV, especially those belonging to the genotype 2, have been difficult to propagate in laboratory cell lines. This study describes the successful isolation of an efficiently growing SAFV-2 strain directly from a stool specimen by standard virological methods. The availability of SAFV isolates that can be propagated to high titers is crucial to the future studies on pathogenesis and epidemiology of these novel human viruses. J. Med. Virol. 84:1497–1500, 2012. © 2012 Wiley Periodicals, Inc.