Adenoviruses (Ads) have demonstrated significant success as replication-deficient (RD) viral vectored vaccines, as well as broad potential across gene therapy and cancer therapy. Ad vectors transduce human cells via direct interactions between the viral fiber knob and cell surface receptors, with secondary cellular integrin interactions. Ad receptor usage is diverse across the extensive phylogeny. Commonly studied human Ad serotype 5 (Ad5), and chimpanzee Ad-derived vector "ChAdOx1" in licensed ChAdOx1 nCoV-19 vaccine, both form primary interactions with the coxsackie and adenovirus receptor (CAR), which is expressed on human epithelial cells and erythrocytes. CAR usage is suboptimal for targeted gene delivery to cells with low/negative CAR expression, including human dendritic cells (DCs) and vascular smooth muscle cells (VSMCs). We evaluated the performance of an RD Ad5 vector pseudotyped with the fiber knob of human Ad serotype 49, termed Ad5/49K vector. Ad5/49K demonstrated superior transduction of murine and human DCs over Ad5, which translated into significantly increased T cell immunogenicity when evaluated in a mouse cancer vaccine model using 5T4 tumor-associated antigen. Additionally, Ad5/49K exhibited enhanced transduction of primary human VSMCs. These data highlight the potential of Ad5/49K vector for both vascular gene therapy applications and as a potent vaccine vector.