Most pathogens access the body through mucosal membranes; therefore it is a high priority in global health to develop vaccines capable of establishing protective immune responses at mucosal sites, as well as systemic sites. However, induction of mucosal immunity by systemic administration of vaccines has proven difficult due to the unique immunological features of the mucosal immune system. Reports showed that intramuscular (i.m.) vaccination of an adenovirus vector (Adv) induces functional and sustainable antigen (Ag)-specific cytotoxic T lymphocytes (CTLs) in gut-mucosal compartments, as well as systemic compartments, in mice and rhesus macaques. Therefore, an Adv has potential as a next-generation mucosal vaccine. Adv-derived nucleic acids, adenoviral genomic DNA and noncoding RNA, trigger innate immune responses through several pathways, resulting in robust production of type I interferons (IFNs). We previously revealed, using Ifnar2−/− mice, that type I IFN signaling is required for induction of gut-mucosal, but not systemic, CTLs following vaccination; however, the molecular mechanism involving type I IFN signaling remains unknown. Here, we found that, in the draining lymph nodes of Ifnar2−/− mice, Th17-polarizing cytokines expression was down-regulated, resulting in the reduction of Ag-specific Th17 cells. Additionally, we revealed that type I IFN signaling was indispensable for induction of Ag-specific Th17 cells in gut mucosa following i.m. Adv vaccination, and that transfer of Th17 cells enhanced the induction of Ag-specific CTLs in gut mucosa, but not in systemic sites. These data suggested that Th17 cells translate systemic type I IFN signaling into gut-mucosal CTL response following i.m. Adv vaccination. We believe that our results will promote the development of advanced mucosal vaccines based on the novel concept of Th17 induction.