Stachyflin is a novel compound having H1 and H2 subtype-specific anti-influenza A virus activity. Stachyflin has no inhibition on H3 subtype influenza A or influenza B viruses. The susceptibility of the reassortant viruses between H1 and H3 subtype influenza A viruses to Stachyflin indicated that its target was virus-encoded hemagglutinin (HA). The results of the timing of Stachyflin addition against in vitro virus infection and virus-mediated hemolysis assay suggested that the drug inhibited the HA-mediated virus-cell fusion process. More directly, Stachyflin interfered with HA conformational change induced by low pH or heat treatment. The effect of Stachyflin could not be eliminated by washing of the Stachyflin-treated virus, which caused very specific virucidal effect. This is a remarkable property among small molecules which inhibit low-pH induced HA conformational change. From these findings, we concluded that the mechanism of Stachyflin action is to inhibit HA conformational change which is necessary for virus-cell membrane fusion. Stachyflin may be used as a tool for a study of molecular mechanism of low-pH induced HA conformational change, and offers potential as a pharmaceutical agent.