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Objective: Influenza complications are mild to serious, and can cause death in some cases. A great deal of attention has been paid in recent years to the development and use of new antiviral compounds to overcome drug resistance in certain strains of the influenza virus and treat the clinical implications. This study aimed to investigate the antiviral effect of punicalagin and its associated mechanism against influenza A (H1N1) virus in vitro. Materials and Methods: the ant-influenza activity of punicalagin was studied in Madin-Darby Canine Kidney (MDCK) cells using influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) using Hemagglutinin assay (HA) and 50% tissue culture infective dose (TCID50). Then, the inhibition of haemagglutination, virucidal activity, inhibitory effect at different times, replication of viral RNA and expression of viral genes were investigated. Results: Punicalagin could inhibit influenza virus infection with 50% inhibitory concentration (IC50) of 3.98 µg/ml and selectivity index (SI) value of 6.1. Punicalagin decreased virus titers with an inhibitory effect on virus hemagglutination (p<0.05). Punicalagin also inhibited viral adsorption. The results of virus RNA replication and viral mRNA (NS1 and HA) expression after treatment with punicalagin showed significant suppression of viral mRNA expression but no effect on replication of viral RNA. Conclusion: The results of the present study indicated that punicalagin was effective against influenza infection most probably via inhibition of haemagglutination activity and virus binding. [ABSTRACT FROM AUTHOR]