Dendritic cells (DC) are central to the initiation of immune responses and various approaches have been used to target vaccines to DC in order to improve immunogenicity. Cannulation of lymphatic vessels allows for the collection of DC that migrate from the skin. These migrating DC are involved in antigen uptake and presentation following vaccination. Human replication-deficient adenovirus (AdV) 5 is a promising vaccine vector for delivery of recombinant antigens. Although the mechanism of AdV attachment and penetration has been extensively studied in permissive cell lines, few studies have addressed the interaction of AdV with DC. In this study, we investigated the interaction of bovine skin-migrating DC and replication deficient AdV-based vaccine vectors. We found that despite lack of expression of CAR and other known adenovirus receptors, AdV readily enters skin-draining DC via actin-dependent endocytosis. Virus exit from endosomes was pH-independent and neutralizing antibodies did not prevent virus entry but did prevent virus translocation to the nucleus. We also show that combining adenovirus with adjuvant increases the absolute number of intracellular virus particles per DC but not the number of DC containing intracellular virus. This results in increased trans-gene expression and antigen presentation. We propose that in the absence of CAR and other known receptors, AdV5-based vectors enter skin-migrating DC using actin-dependent endocytosis which occurs in skin-migrating DC and its relevance to vaccination strategies and vaccine vector targeting is discussed.