Mx proteins are interferon induced, antiviral proteins, expressed in response to treatment with double stranded RNA or virus infection. Here we report the cloning, sequencing, and antiviral property of three forms of Mx genes, MxI, MxII, and MxIII from grouper (Epinephelus coioides). Multiple comparison of grouper Mx amino acid sequences revealed the conservation of Mx putative GTP-binding domain, dynamin family signature, and leucine zipper motif. We have established a new cell line, grouper brain 3 (GB3), and prepared stable clones expressing Flag-epitope tagged grouper MxI, MxII, and MxIII. Immunostaining shows that all the three grouper Mx proteins are localized in the cytoplasm. To examine the antiviral activity of grouper Mx proteins, these stable clones were infected by a nodavirus, yellow grouper nervous necrosis virus (YGNNV), and the results showed that all the three Mx isoforms have the efficiency of reducing the titre of virus from 10- to 100-fold. Moreover, through immunocytochemistry we demonstrated that Mx protein can inhibit the YGNNV propagation in GB3 cells. Taken together, this study demonstrates that grouper Mx proteins have efficient inhibitory activity against nodavirus, the most endangered virus of fish, and this information would be helpful to design effective DNA vaccines that can confer an early non-specific antiviral protection.