Adenovirus vectors have great potential in cancer gene therapy. Targeting of cancer-testis (CT) antigens, which are specifically presented at the surface of tumor cells by human leukocyte antigen (HLA) class I molecules, is an attractive option. In this study, a single-chain T-cell receptor (scTCR) directed against the CT antigen melanoma-associated antigen (MAGE)-A1 in complex with the HLA class I molecule of haplotype HLA-A1 is fused with the C terminus of the adenovirus minor capsid protein IX. Propagation of a protein-IX (pIX)-gene-deleted human adenovirus 5 (HAdV-5) vector on cells that constitutively express the pIXscTCR fusion protein yielded viral particles with the pIXscTCR fusion protein incorporated in their capsid. Generated particles specifically transduced melanoma cell lines expressing the HLA-A1/MAGE-A1 target complex with at least 10-fold higher efficiency than control viruses. Whereas loading of HLA-A1-positive cells with MAGE-A1 peptides leads to enhanced transduction of the cells, the efficiency of virus transduction is strongly reduced if the HLA-A1 molecules are not accessible at the target cell. Taken together, these data provide proof of principle that pIXscTCR fusions can be used to target HAdV-5 vectors to tumor cells expressing intracellular CT antigens.