Generation of antigen‐specific CD8+ T cell responses is considered optimal for an effective immunotherapy against cancer. In this study, we provide a proof of principle that in vitro observed diminished CD8+ T cell response provided a strong in vivo tumor protection. Immunization with an adenovirus vaccine containing ovalbumin (OVA) gene (Ad5‐OVA) strongly induces antigen‐specific CD8+ T cell responses measured in vitro using various immunological assays. However, in an attempt to augment the antigenic CD8+ T cell response, coinjection of a TLR9 agonist CpG ODN with the viral vaccine unexpectedly reduced the CD8+ T cell responses measured in vitro but provided a remarkably enhanced tumor protection compared to the CD8+ T cell response generated by Ad5‐OVA vaccine alone. Interestingly, despite reduced ex vivo/in vitro CD8+ T cell responses following Ad5‐OVA+CpG immunization, immunodepletion studies revealed that the augmented anti‐tumor immunity was primarily dependent on CD8+ T cells. The magnitude and effector function of anti‐OVA CD8+ T cells remain low following primary and secondary antigenic challenge, presenting a dichotomy between in vitro CD8 T cell responses and in vivo anti‐tumor immunity. To examine the impact of CpG ODN, we observed that presence of CpG suppresses the CD8+ T cell proliferation both in vitro and in vivo. These data demonstrate that coadministration of adenovirus vaccine with a TLR9 agonist can generate potentially effective tumor‐reactive CD8+ T cells in vivo. In addition, the results indicate that widely used standard immune parameters may not predict the vaccine efficacy containing a TLR9 agonist as adjuvant. © 2007 Wiley‐Liss, Inc.