The immunosuppressive drug cyclosporin A (CsA) has inhibitory effects on the replication of several viruses. The antiviral effects are through targeting the interaction between viral proteins and host factor cyclophilin A (CypA). CypA has been identified to interact with influenza A virus M1 protein and impair the early stage of the viral life cycle. In order to identify the effect of CsA on influenza virus replication, a CypA-depleted 293T cell line, which was named as 293T/CypA-, was constructed. The cytopathic effect (CPE) assay and the growth curve results indicated that CsA specifically suppressed the influenza A virus replication in a dose-dependent manner. CsA treatment had no effect on the viral genome replication and transcription but selectively suppressed the viral proteins expression. Further studies indicated that CsA could impair the nuclear export of viral mRNA in the absence of CypA. In addition, the antiviral activity of CsA was independent of calcineurin signaling. Finally, CsA could enhance the binding between CypA and M1. The above results suggested that CsA inhibited the replication of influenza A virus through CypA-dependent and -independent pathways.