Application of adenovirus vectors (Adv) in metastatic cancer treatment is limited. We previously demonstrated that covalent conjugation of polyethleneglycol (PEG) to Adv enhances therapeutic effects and decreases toxic side-effects after systemic administration, but the level of immune response to PEGylated Adv (PEG-Ad) was not examined. Here, we examined the effect of PEGylation of Adv on the production of anti-Adv antibodies and antitumor response. We constructed a set of PEG-Ad using 5-kDa PEG, with modification rates of 30%, 45% and 90%. After systemic administration of Advs to rats, we examined the level of anti-Adv immunoglobulin (Ig)G and IgM in serum. The levels of anti-Adv IgG and anti-Adv IgM in rats treated with unmodified Adv were higher than those in control group. Rats treated with PEG-Ad that had a 90% modification rate showed lower level of anti-Adv IgG and anti-Adv IgM than those treated with unmodified Adv, whereas rats treated with PEG-Ad that had a 30% or 45% modification rate showed a similar level of anti-Adv IgG and IgM to those treated with unmodified Adv. Systemic administration of PEG-Ad that had a 90% modification rate, and expressed tumor necrosis factor-alpha, significantly reduced the number of metastatic colonies in the lung compared to unmodified Adv, with negligible side effects. These results suggest that systemic administration of PEG-Ad with an appropriate PEG modification rate has the potential to reduce the production of antibodies against Adv and increase the therapeutic response against metastatic cancer.