CD8⁺ T-cell immunity has been shown to play an important role in the protective immune response against Encephalitozoon cuniculi. Although earlier studies suggest that dendritic cells (DC) are important for the induction of this response, the factors responsible for initiation of the dendritic cell response against this pathogen have not been evaluated. In the current study, we demonstrate that E. cuniculi infection causes strong Toll-like receptor 4 (TLR4)-dependent dendritic cell activation and a blockade of this molecule reduces the ability of DC to prime an antigen-specific CD8⁺ T-cell response. Pretreatment of DC with anti-TLR4 antibody causes a defect in both in vitro and in vivo CD8⁺ T-cell priming. These findings, for the first time, emphasize the contribution of TLR4 in the induction of CD8⁺ T-cell immunity against E. cuniculi infection.