Substituting the coat proteins of adenoviral vector serotype 5 (Ad5) can alter vector tropism and circumvent vector neutralization. Here we report that an Ad5 vector carrying a part of the fiber molecule of human subgroup B adenovirus serotype 35 (Ad5.Fib35) transduces cultured human dendritic cells (DC) and circulating myeloid derived DC with approximately 10-fold greater efficiency than Ad5 in vitro. The improved DC transduction results in increased T-cell activation ex vivo. In vivo however, immunogenicity of the vectors in mice and non-human primates did not correlate with in vitro DC tropism. Ad5.Fib35 was less immunogenic in monkeys than Ad5, despite the improved primate DC tropism of Ad5.Fib35. In mice with high Ad5 vector-specific immunity, Ad5.Fib35 showed no significant difference in anti-insert immunity over Ad5 indicating that fiber exchange alone does not evade pre-existing Ad5 immunity. We thus conclude that, for ex vivo vaccination, Ad5.Fib35 shows promise as vector for loading of DC but is unable to circumvent anti-Ad5 immunity limiting its in vivo utility.