Acetylated microtubules (AcMTs), a post-translationally modified form of microtubules, promote polarized protein transport. Here we report that influenza A virus (IAV) induces the acetylation of microtubules in epithelial cells. By employing specific inhibitors and siRNA we demonstrate Rho GTPase-mediated downregulation of tubulin deacetylase activity in IAV-infected cells, resulting in increased tubulin acetylation. Further, we demonstrate that depolymerization/deacetylation or enhanced acetylation of microtubules decreased or increased, respectively, the release of virions from infected cells. IAV assembly requires the polarized delivery of viral components to apical plasma membrane. Our findings suggest the potential involvement of AcMTs in polarized trafficking of IAV components. © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.