Abstract: To investigate the functional properties of interferon, alpha-inducible protein 27 like 2 a (ifi27l2a) and the differences in the mechanisms of anti-grass carp reovirus (GCRV) between C. idella and S. curriculus, this study applied cloning, multiple sequence alignment, prokaryotic expression, qPCR and other methods to compare the sequence characteristics, expression characteristics and antiviral function of ifi27l2a between C. idella and S. curriculus. The results showed that the open reading frames of ifi27l2a in C. idella and S. curriculus are both 288 bp, encoding 95 amino acids, with high sequence homology, but there are differences in the β-sheets. Ifi27l2a is widely distributed in various tissues of C. idella and S. curriculus. After GCRV infection, it is significantly induced in the liver, spleen, and kidney of C. idella, but only in the heart of S. curriculus, and the induction expression in C. idella is much higher than that in S. curriculus. Meanwhile, in the kidney cell lines of C. idella and S. curriculus, the induced expression level of ifi27l2a in CIK at 12 h post GCRV infection was significantly higher than that in SCK, which was similar to the results of the individual, suggesting that the differences in the expression pattern and the induced expression of ifi27l2a might be an important factor in the differences in the resistance ability between C. idella and S. curriculus. To compare the antiviral function of the IFI27L2A protein in C. idella and S. curriculus, the recombinant proteins rScIFI27L2A and rCiIFI27L2A were successfully prepared in this experiment. The results of virus infection and protein incubation experiments confirmed that both can effectively inhibit GCRV replication. It is worth mentioning that the recombinant protein rScIFI27L2A significantly inhibits the IFN response induced by GCRV in SCKs, while the recombinant protein rCiIFI27L2A significantly enhances the IFN response in CIKs, indicating that there are significant differences in the antiviral regulatory mechanisms of IFI27L2A in C. idella and S. curriculus. Our research results provide clues for the molecular mechanism of resistance difference to GCRV in C. idella and S. curriculus, and provide theoretical references for the development of antiviral drugs for fish and in-depth research on fish antiviral mechanisms.