Vaccination with the live vaccine strain (LVS) or natural infection with Francisella tularensis confers long-lasting protection against re-infection, and it is almost exclusively dependent on cell-mediated immunity. The extent, if any, to which vaccine-induced and infection-induced immunity differ in quality and persistence remains unclear. In this study, we compared human immune responses resulting from LVS vaccination and tularemia infection. Peripheral blood mononuclear cells from vaccinees and convalescent patients were recall-stimulated and analyzed for proliferation, characterization of cytokine-producing memory T cells, and cytokine secretion. Both vaccination and infection elicited robust antigen-specific T-cell activation, accompanied by coordinated cytokine release. Intracellular staining identified multifunctional CD4 and CD8 T cells after both vaccination and infection. Characterization of memory subsets revealed that vaccination primarily induced transitional and effector memory populations, while natural infection generated broader and more persistent responses, including activation of terminally differentiated TEMRA (Terminal Effector Memory T cells re-expressing CD45RA) cells and higher intracellular expression of MIP-1β by CD4 and CD8 cells. These findings demonstrate that both LVS vaccination and natural infection induce efficacious immune responses to F. tularensis, as evidenced by the presence of long-lasting, multifunctional T cells, but with partially distinct memory profiles. The broader and more durable responses after natural infection highlight areas of relevance to understand the efficacy of existing and future tularemia vaccines.