HDAC6 is a tubulin deacetylase involved in many cellular functions related to cytoskeleton dynamics including cell migration or autophagy. In addition, HDAC6 affects antigen-dependent CD4(+) T cell activation. In this study, we show that HDAC6 contributes to the cytotoxic function of CD8(+) T cells. Immunization studies revealed defective cytotoxic activity in vivo in the absence of HDAC6. Adoptive transfer of wild-type or hdac6(-/-) CD8(+) T cells to rag1(-/-) mice demonstrated specific impairment in CD8(+) T cell responses against vaccinia infection. Mechanistically, HDAC6-deficient cytotoxic T lymphocytes (CTL) showed defective in vitro cytolytic activity related to altered dynamics of lytic granules, inhibited kinesin 1 - dynactin mediated terminal transport of lytic granules to the immune synapse and deficient exocytosis, but not target cell recognition, TCR activation or IFNγ production. Our results establish HDAC6 as an effector of the immune cytotoxic response by affecting the dynamics, transport and secretion of lytic granules by CTL.