The unique ability of human adenovirus serotype 5 (Ad5) to accomplish efficient transduction has allowed the use of Ad5-based vectors for a range of gene therapy applications. Several strategies have been developed to alter tropism of Ad vectors to achieve a cell-specific gene delivery by using fiber modifications via genetic incorporation of targeting motifs. In this study, we have explored the utility of novel anti-human carcinoembryonic antigen (hCEA) single variable domains derived from heavy chain (VHH) camelid family of antibodies to achieve targeted gene transfer. To obtain anti-CEA VHHs, we produced a VHH-display library from peripheral blood lymphocytes RNA of alpacas at the peak of immune response to the hCEA antigen (Ag). We genetically incorporated an anti-hCEA VHH into a de-knobbed Ad5 fiber-fibritin chimera and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of target cells. We report that the anti-hCEA VHH used in this study retains Ag recognition functionality and provides specificity for gene transfer of capsid-modified Ad5 vectors. These studies clearly demonstrated the feasibility of retargeting of Ad5-based gene transfer using VHHs.