The emergence of highly contagious influenza A virus strains, such as the new H1N1 swine influenza, represents a serious threat to global human health. Efforts to control emerging influenza strains focus on surveillance and early diagnosis, as well as development of effective vaccines and novel antiviral drugs. Herein we document the anti-influenza activity of the anti-infective drug nitazoxanide and its active circulating-metabolite tizoxanide and describe a class of second generation thiazolides effective against influenza A virus. Thiazolides inhibit the replication of H1N1 and different other strains of influenza A virus by a novel mechanism: they act at post-translational level by selectively blocking the maturation of the viral hemagglutinin at a stage preceding resistance to endoglycosidase H digestion, thus impairing hemagglutinin intracellular trafficking and insertion into the host plasma membrane, a key step for correct assembly and exit of the virus from the host cell. Targeting the maturation of the viral glycoprotein offers the opportunity to disrupt the production of infectious viral particles attacking the pathogen at a level different from the currently available anti-influenza drugs. The results indicate that thiazolides may represent a new class of antiviral drugs effective against influenza A infection.