Replication-incompetent adenovirus (Ad) vectors have gained attention as gene delivery vehicles. Theoretically, no Ad genes should be expressed following transduction; however, Ad genes are expressed from the vector genome, leading to induction of cellular immunity against Ad proteins and Ad protein-induced toxicity. To suppress the leaky expression of Ad genes, a microRNA (miRNA)-regulated gene expression system was utilized. We developed novel Ad vectors by incorporating targeted sequences of miR-122a or miR-142-3p, which exhibit liver- or spleen-specific expression, respectively, in the 3′-untranslated region (UTR) of the E2A, E4, or pIX genes. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional human embryonic kidney 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold in an miRNA-dependent manner, compared with a conventional Ad vector, by the insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a-targeted sequences into the 3′-UTR of the E4 gene (Ad-E4-122aT) expressed 1.5- to 34-fold higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver compared with a conventional Ad vector. miR-122a-mediated suppression of E4 gene expression in the liver significantly reduced the hepatotoxicity that an Ad vector causes via both adaptive and non-adaptive immune responses. Ad-E4-122aT would be a promising framework for efficient gene delivery due to its ability to mediate higher and longer-term transgene expression and lower hepatotoxicity than a conventional Ad vector.