It has been shown that host protein cyclophilin A (CypA) played a key role in the replication of several viruses. In order to address the function of Mandarin fish CypA (SC-CypA) in infectious spleen and kidney necrosis virus (ISKNV) replication, the full ORF of SC-CypA was first cloned and characterized. The ORF of SCCypA encoded a polypeptide of 164 amino acids with calculated molecular weight of 17.59 ku. The deduced amino acid sequences of the SC-CypA shared highly conserved structures with CypAs from other species, indicating that SC-CypA should be a new member of the CypA family. Cyclosporin A (CsA) is a specific inhibitor of CypA. ISKNV replication was inhibited by the addition of CsA in a manner of dose dependence. CsA could effectively inhibit the replication of ISKNV at different time points after ISKNV infection, and the strongest inhibitory effect was observed at 72 h poi. The addition of CsA could inhibit the mRNA expressions of IL-1β, IL-8, IL-18 and ISG15 in CPB cells after the infection of ISKNV. These suggest that SC-CypA plays a key role in the ISKNV replication, and CsA could effectively inhibit the replication of ISKNV.