Tumor-specific adenoviral vectors comprise a fruitful gene-based diagnostic imaging and therapy research area for advanced stage of cancer, including metastatic disease. However, clinical translation of viral vectors has encountered considerable obstacles, largely due to host immune responses against the virus. Here, we explored the utilization of an immunosuppressant, rapamycin, to circumvent the anti-adenovirus immunity in immunocompetent murine prostate cancer models. Rapamycin diminished adenoviral-induced acute immune response by inhibiting NF-κB activation; it also reduced the scale and delayed the onset of inflammatory cytokine secretion. Further, we found that rapamycin abrogated anti-adenovirus antibody production and retarded the function of myeloid cells and lymphocytes that were activated upon viral administration in pre-immunized hosts. Thus, the co-administration of rapamycin prolonged and enhanced adenovirus-delivered transgene expression in vivo, and thereby augmented the imaging capability of adenoviral vectors in both bioluminescent and positron emission tomography modalities. Furthermore, we showed that despite an excellent response of cancer cells to a cytotoxic gene therapeutic vector in vitro, only minimal therapeutic effects were observed in vivo in pre-immunized mice. However, when we combined gene therapy with transient immunosuppression, complete tumor growth arrest was achieved. Overall, transient immunosuppression by rapamycin was able to boost the diagnostic utility and therapeutic potentials of adenoviral vectors.