This study was conducted to evaluate the regulation mechanism of influenza virus replication following treatment of Madin-Darby canine kidney cells with the soy isoflavone daidzein. We performed comparative qualitative and quantitative analyses of lipid peroxide between mock-infected and virus-infected cells treated with or without daidzein, as it had been reported that daidzein was an antioxidant and lipid peroxide levels increased upon virus infection. Contrary to our belief, lipid peroxides were not elevated in virus-infected cells and no decrease in lipid peroxides was observed in daidzein-treated cells. In daidzein-treated cells, 5-hydroxyeicosatetraenoic acid, the 5-lipoxygenase product derived from arachidonate, was significantly elevated compared to other lipid peroxides. Zileuton (5-lipoxygenase inhibitor) and 5-lipoxygenase knockdown reduced the daidzein-induced antiviral effect. Moreover, virus replication was regulated by treatment with 5-hydroperoxyeicosatetraenoic acid, a precursor of 5-hydroxyeicosatetraenoic acid and 5-lipoxygenase primary product. These results suggest that daidzein regulates virus replication via signal transduction through 5-lipoxygenase products.