A major obstacle to the clinical application of systemic adenoviral gene replacement therapy is the host innate immune response. Although recent studies have attempted to characterize the cellular basis for this response to systemically administered helper-dependent adenoviral vector (HD-Ad), the underlying molecular components of the innate immune repertoire required to recognize the viral vector have yet to be identified. Here, we show that primary macrophages can sense HD-Ad vectors via the Toll-like Receptor 9 (TLR9) and respond by increasing pro-inflammatory cytokine secretion. Moreover, TLR9 sensing is involved in the rapid innate immune response to HD-Ad in vivo. TLR9 deficiency attenuates the innate immune response to HD-Ad, whereas TLR9 blockade reduces the acute inflammatory response after intravenous injection of the vector. Moreover, HD-Ad upregulates TLR9 gene expression independent of TLR9 function, suggesting that additional innate signaling pathways work cooperatively with TLR9. The identification of the components of the innate immune response to adenovirus will facilitate the development of combinatorial therapy directed at increasing the maximal tolerated dose of systemically delivered adenoviral vectors.