HER-2/neu transgene-modified dendritic cell (DC)-based vaccines are potent at eliciting HER-2/neu-specific antitumor immunity. In this study, we constructed a recombinant adenovirus (RGD)AdVneu with fiber gene modified by RGD insertion into the viral knob's H1 loop. We transfected DCs with (RGD)AdVneu, and assessed/compared HER-2/neu-specific humoral and cytotoxic T lymphocyte (CTL) responses and antitumor immunity derived from the original AdVneu-transfected DCs (DCneu1) and (RGD)AdVneu-transfected DCs (DCneu2). We demonstrated that DCneu2 displayed increased HER-2/neu expression by 8.3-fold compared to DCneu1. We also demonstrated that DCneu2 vaccination induced stronger HER-2/neu-specific humoral and CTL immune responses than DCneu1 vaccination. DCneu2 vaccination protected all the mice from HER-2/neu-expressing Tg1-1 tumor cell challenge in wild-type FVB/NJ mice, compared to a partial protection in DCneu1-immunized mice. In addition, DCneu2 vaccination also significantly delayed tumor growth than DCneu1 immunization (P<0.05) in Tg FVBneuN mice. Three immunizations of DCneu2 starting at the mouse age of 2 months also significantly delayed breast cancer development in Tg mice compared to DCneu2 vaccine (P<0.05). Importantly, DCneu2 vaccine reduced breast carcinogenesis by 9% in Tg mice with self HER-2/neu tolerance. Therefore, vaccination of fiber-modified adenovirus-transfected DCs to enhance expression of tumor antigens such as HER-2/neu is likely representative of a new direction in DC-based vaccine of breast cancer.