Recombinant adenoviruses have been used widely for gene therapy due to their high transduction efficiency in vivo. However, the attendant innate immune response to adenoviral vectors has limited their applications for in vivo gene therapy. Recent studies have shown that adenoviruses activate the innate immunity through both Toll-like receptor–dependent (TLR-dependent) and TLR–independent pathways, leading to the production of type I interferons (IFNs) and other inflammatory cytokines. Furthermore, type I IFNs play a pivotal role in innate immune elimination of adenoviral vectors in vivo. It remains to be defined how type I IFNs regulate innate immune clearance of adenoviral vectors. In this study, we showed in vivo that natural killer (NK) cells were activated and accumulated in the liver upon intravenous administration of adenoviral vectors, leading to the loss of adenoviral genome and the reduction of transgene expression. We further demonstrated that type I IFNs were critical for the activation of NK cells. This was achieved by direct action of type I IFNs on NK cells. Overall, our observations reveal a critical role for type I IFN–dependent NK cell activation in innate immune elimination of adenoviral vectors in vivo and may help design effective strategies to improve the outcome of adenovirus-mediated gene therapy.