Adenoviral vectors are commonly used for liver-directed gene therapy following systemic administration owing to their strong propensity for hepatocyte transduction. However, many disease applications would benefit from the delivery of adenoviruses to alternate tissues via this route. Research has thus focused on stripping the virus of native hepatic tropism in conjunction with modifying virus capsid proteins to incorporate novel tropism. Recently, the KO1S* adenovirus serotype 5 fiber mutant, devoid of both coxsackie and adenovirus receptor binding in the fiber knob domain and mutated at the putative heparan sulphate proteoglycan binding site in the fiber shaft, was shown to possess strikingly poor hepatic tropism in mice, rats, and non-human primates. Thus, it is an ideal candidate for retargeting strategies. We therefore assessed the ability of peptide-modified KO1S* fibers to retarget adenovirus. Peptide insertions were well tolerated and virions produced to high titers. However, expected retargeting at the level of transduction was not observed, despite cell-binding studies showing enhanced vector targeting at the cell surface. Cy3 labeling studies showed retarded trafficking of S*-containing fibers. Taken together, our data demonstrates that KO1S* mutant fibers are ineffective for cell retargeting strategies.