Abstract: AIM: To elucidate the inhibitory mechanism of baicalein on influenza A/FM1/1/47 (H1N1) virus in cell culture. METHODS: The inhibition of baicalein on influenza virus infection was investigated using a crystal violet staining assay. Three infection protocols were used to study the inhibitory effects of baicalein on different stages of the replication cycle of the influenza virus. Viral M1 mRNA expression was compared between drug-treated and untreated infected cells to determine the inhibitory effect of baicalein on viral mRNA synthesis using RT-PCR. RESULTS: Both the A/FM1/1/47 (H1N1) and A/Beijing/32/92 (H3N2) influenza viruses were inhibited with different concentrations of baicalein in a dose-dependent manner. Treatment with baicalein after viral inoculation to MDCK cells resulted in stronger inhibitory effects compared with treatments prior to, and at the same time, as viral inoculation. Furthermore, no significant difference was observed between the viral M1 mRNA synthesis in the baicalein-treated group and the untreated group at 3 hpi (hours post-infection), compared to a clear decrease of viral mRNA synthesis at 6 and 9 hpi. CONCLUSION: This study has identified a novel inhibitory mechanism of baicalein on influenza A/FM1/1/47 (H1N1) virus via interference with mid-late mRNA synthesis in cell culture.