Recent studies have generated interest into the function of human adenovirus serotype 5 (HAd-V5) hexon: factor X (FX)-binding and subsequent hepatocyte transduction and interaction with the immune system. Here, we retargeted adenovirus serotype 5 vectors, ablated for FX interaction, by replacing amino acids in hexon HVR7 with RGD-4C or inserting the peptide into the fiber HI loop. These genetic modifications in the capsid were compatible with virus assembly, and could efficiently retarget transduction of the vector via the αvβ3/5 integrin-mediated pathway, but did not alter immune recognition by pre-existing human neutralising anti-HAdV-5 antibodies or by natural antibodies in mouse serum. Thus, FX-binding ablated HAdV-5 can be retargeted but remain sensitive to immune-mediated attack. These findings further refine HAd-V5 based vectors for human gene therapy and inform future vector development.