Grass carp reovirus (GCRV) is the causative agent of grass carp hemorrhagic disease, which has detrimental effects on the grass carp aquaculture industry. There are four known genotypes of GCRV and strains of different GCRV genotypes differ greatly. In this study, the diversity of the protein VP6 from different GCRV stains and the effect of genotype GCRV 096 on replication was investigated in CIK cells. Our results showed that the VP6 protein of GCRV 096 (genotype Ι) exhibited limited homology to that of GCRV GD108 (genotype Ш), with few residues conserved in predicted protein-protein interaction domains. GCRV 096 VP6 protein was expressed and purified and an antiserum against it was characterized. Addition of purified VP6 protein or antiserum to culture media of CIK cells inhibited the replication of GCRV 096 in these cells. In contrast, replication of GCRV GD108 (genotype Ш) was not affected in CIK cells under the same condition. Overall, our results indicated that the protein VP6 and VP6 antiserum did not provide cross-protection against GCRV strains and this can be attributed to differences among GCRV genotypes. It will be important to consider multiple GCRV genotypes in the development of effective GCRV vaccines and other therapies against grass carp hemorrhagic disease. In addition, bioinformatics analysis also suggested that the protein VP6 may have a role in the process of GCRV infection. This study lays the foundation for the prevention of grass carp hemorrhagic disease and further detailed studies on the pathogenesis of GCRV. Keywords: Grass carp reovirus (GCRV), Protein VP6, Replication, Functional analysis