The aim of this study was to evaluate the usefulness of replicating but non disseminating adenovirus vectors (AdVs) as vaccine vector using human rotavirus (HRV) as a model pathogen. HRV VP7, VP4, or VP4Delta (N-terminal 336 amino acids of VP4) structural proteins as well as the VP4Delta::VP7 chimeric fusion protein were expressed in mammalian cells when delivered with the AdVs. A preliminary experiment demonstrated that VP4Delta was able to induce a HRV-specific IgG response in BALB/c mice inoculated intramuscularly with AdVs expressing the rotaviral protein. Moreover, an AdV-prime/plasmid DNA-boost regimen of vectors resulted in VP4Delta-specific antibody (Ab) titers ~4 times higher than those obtained from mice immunized with AdVs alone. Subsequently, the various HRV protein-encoding AdVs were compared using the AdV-prime/plasmid DNA-boost regimen. Higher IgG and IgA responses to HRV were obtained when VP4Delta::VP7 fusion protein was used as an immunogen as compared to VP7 or VP4 alone or to a mix of both proteins delivered independently by AdVs. A synergetic effect in terms of Ab was obtained with VP4Delta::VP7. In conclusion, this study demonstrated for the first time the suitability of using replicating but non disseminating AdVs as vaccine vector and the VP4Delta::VP7 fusion protein as an immunogen for vaccination against HRV.