Despite the advantages of using adenoviral vectors for specific antigenic gene delivery in the development of antigen-presenting cell (APC)-based vaccines, the lack of the coxsackievirus-adenovirus receptor (CAR) on APCs limits the use of adenoviral vectors for in vitro gene delivery. In this study, we used a recombinant adapter protein, CFm40L, which consists of the ectodomain of CAR genetically fused to the ectodomain of CD40 ligand (CD40L) via a trimerization motif, to target Her-2/neu- or human papillomavirus 16 (HPV16) E6/E7-encoding adenoviruses to CD40 on dendritic cells (DCs) and B cells. Targeting CD40 enabled the enhancement of tumor antigen delivery and simultaneous activation of APCs via the CD40-CD40L interaction. We found that these transduced DCs and B cells substantially enhanced the CTL response against human Her-2/neu- and HPV16 E6/E7-expressing tumors, resulting in significant inhibition of tumor growth in a murine tumor model. In addition, the use of the CFm40L adapter protein in combination with gemcitabine treatment allowed for a successful immune response against a self-tumor antigen, murine Her-2/neu. Our results suggest that targeting adenovirus to APCs via CD40, using CFm40L, represents a great improvement in anticancer cellular vaccines.