Protective T-cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II-associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4(+) T cells. Here, we investigated if Ii could similarly activate human CD8(+) T cells when used as a vehicle for cytotoxic T-cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART-1 or CD20, coprecipitated with HLA-A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA-A*02:01-positive cells expressing CLIP-replaced Ii efficiently activated Ag-specific CD8(+) T cells in a TAP- and proteasome-independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART-1 or IiCD20 primed naïve CD8(+) T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.