Adenovirus (Ad) is being explored for use in the prevention and treatment of a variety of infectious diseases and cancers. Ad with a deletion in early region 3 (DE3) provokes a stronger immune response than Ad with deletions in early regions 1 and E3 (DE1/DE3). The DE1/DE3 Ads are more popular because they can carry a larger transgene and, because of the deleted E1 (E1A and E1B), are perceived safer for clinical use. Ad with a deletion in E1B55K (DE1B55K) has been in phase 3 clinical trials for use in cancer therapy in the United States and has been approved for use in head and neck tumor therapy in China, demonstrating that Ads containing E1A are safe for clinical use. We have shown previously that DE1B55K Ad, even while promoting lower levels of an inserted transgene, promoted levels of transgene-specific immune responses similar to those of a DE3 Ad. Products of the Ad early region 4 (E4) limit the ability of cells to mount an innate immune response. Using this knowledge, we deleted Ad E4 open reading frames 1 (E4orf1) to E4orf4 (E4orf1-4) from the DE1B55K Ad. Here, we show that innate cytokine network genes are elevated in DE4 Ad-infected cells beyond that of DE3 Ad-infected cells. Furthermore, in immunized mice, the IgG2a subclass was favored, as was the IgG1 subclass, in immunized nonhuman primates. Thus, Ad E4 impacts immune responses in cells, immunized mice, and immunized nonhuman primates. These Ads may offer advantages that are beneficial for clinical use. IMPORTANCE Adenovirus (Ad) is being explored for use in the prevention and treatment of a variety of infectious diseases and cancers. Here, we provide evidence in cells, mice, and nonhuman primates supporting the notion that Ad early gene products limit specific immune responses. Ad constructed with deletions in early genes and expressing HIV envelope protein was shown to induce greater HIV-specific cellular immune responses and higher-titer antibodies than the parental Ad with the early genes. In addition to eliciting enhanced immunity, the deleted Ad possesses more space for the insertion of additional or larger transgenes needed for targeting other infectious agents or cancers.