Adenoviral (Ad) vectors have proven to be important tools for gene and cell therapy, although some issues still need to be addressed, such as undesired interactions with blood components and off-target sequestration that ultimately hamper efficacy. In the past years, several organic and inorganic materials have been developed to reduce immunogenicity and improve biodistribution of Ad vectors. Here we investigated the influence of the functionalization of 14 nm PEGylated gold nanoparticles (AuNPs) with quaternary ammonium groups and an arginine-glycine-aspartic acid (RGD)-motif on the uptake and biodistribution of Ad vectors. We report the formation of Ad@AuNPs complexes that promote cell attachment and uptake, independently of the presence of the coxsackievirus and adenovirus receptor (CAR) and αvβ3 and αvβ5 integrins, significantly improving transduction without limiting Ad bioactivity. Besides, the presence of the RGD peptide favors tumor targeting and decreases Ad sequestration in the liver. Additionally, tumor delivery of a coated Ad vector expressing the human sodium iodide symporter (hNIS) by mesenchymal stem cells induces increased accumulation of radioactive iodine (131I) and tumor volume reduction compared to naked Ad-hNIS, highlighting the promising potential of our coating formulation in cancer gene therapy. Statement of significance: Modification of adenoviral vectors with lipids and polymers can reduce interactions with blood components and increase tumor accumulation; however, increased toxicity and reduced transduction efficiency were indicated. Coating with gold nanoparticles has proven to be a successful strategy for increasing the efficiency of transduction of receptor-defective cell lines. Here we explore the contribution of cell surface receptors on the mechanisms of entry of Ad vectors coated with gold nanoparticles in cell lines with varying degrees of resistance to infection. The enhancement of the anti-tumoral effect shown in this work provides new evidence for the potential of our formulation.