Mx proteins are interferon-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses. We previously demonstrated that porcine Mx1 protein (poMx1) inhibited the replication of classical swine fever virus (CSFV), an economically important Pestivirus, and that mouse Mx1 did so as well. It is unknown why the nucleus-localizing mouse Mx1 inhibits CSFV replication which occurs in the cytoplasm. To the end, we assessed the anti-CSFV actions of wild type mouse Mx1 and seven previously reported mutants (K49A, G83R, A222V, A516V, G540E, R614E and ΔL4) and identified the molecular mechanism of R614E action against CSFV replication. A series of experiments revealed that mmMx1 (R614E) mutant reposted to the cytoplasm and interacted with the CSFV nucleocapsid protein (Core), thereby inhibiting viral replication. These findings broaden our understanding of the function of Mx protein family members against CSFV and suggest that the relative conservation of Mx1 among species is the basis of broad-spectrum antiviral properties.