Human adenoviruses have a great potential as anticancer agents. One strategy to improve their tumor-cell specificity and anti-tumor efficacy is to include tumor-specific targeting ligands in the viral capsid. This can be achieved by fusion of polypeptide-targeting ligands with the minor capsid protein IX. Previous research suggested that protein IX-mediated targeting is limited by inefficient release of protein IX-fused ligands from their cognate receptors in the endosome. This thwarts endosomal escape of the virus particles. Here we describe that the targeted transduction of tumor cells is augmented by a cathepsin-cleavage site between the protein IX anchor and the HER2/neu-binding ZH Affibody molecule as ligand. The cathepsin-cleavage site did not interfere with virus production and incorporation of the Affibody molecules in the virus capsid. Virus particles harboring the cleavable protein IX-ligand fusion in their capsid transduced the HER2/neu-positive SKOV-3 ovarian carcinoma cells with increased efficiency in monolayer cultures, three-dimensional spheroid cultures and in SKOV-3 tumors grown on the chorioallantoic membrane of embryonated chicken eggs. These data show that inclusion of a cathepsin-cleavage sequence between protein IX and a high-affinity targeting ligand enhances targeted transduction. This modification further augments the applicability of protein IX as an anchor for coupling tumor-targeting ligands.