Abstract Tick-borne encephalitis (TBE) is a viral infection of the CNS caused by TBE virus. With no specific treatment available, the only protection is a formalin-inactivated whole virus vaccine. Primary immunization with European TBE vaccines, as recommended by the manufacturers, consists of three vaccine doses administered within a 1-y period. Protection from vaccination is believed to be mediated by Abs, yet T cells may also have a protective role. We set out to characterize the human CD4+ T cell response throughout primary TBE immunization. The responses were evaluated before vaccination and 1 mo after each vaccine dose. A heterogeneous magnitude of CD4+ T cell–mediated memory responses was observed in regard to lymphoblast expansion and cytokine production (IFN-γ, IL-2, and TNF), with the highest median magnitude detected after the second dose of vaccine. Stimulation with an overlapping peptide library based on structural TBE virus proteins E and C revealed that CD4+ T cells concomitantly producing IL-2 and TNF dominated the responses from vaccinees after each vaccine dose, whereas a control cohort of TBE patients responded mainly with all three cytokines. CD107a expression was not upregulated upon peptide stimulation in the vaccinees. However, CD154 (CD40L) expression on cytokine-positive memory CD4+ T cells significantly increased after the second vaccine dose. Taken together, TBE vaccination induced CD4+ T cell responses dominated by IL-2 and TNF production together with CD154 upregulation and a lower IFN-γ response compared with TBE patients. This response pattern was consistent after all three doses of TBE vaccine.