Adenovirus vectors are known to induce certain genes and impact innate response networks, but a broad understanding of this process and its mechanisms is currently lacking. For this reason, we chose to investigate and characterize Ad innate immunity using homogeneous, primary MEF cells replete with all the elements of the pathogen-sensing Toll-Like Receptor (TLR) pathway. By using an array-based approach to maximally define transcriptome changes induced upon Ad vector infection, we discovered that Ad infection induces a potent gene and transcription factor network response. This response is characterized by significant changes in the expression of genes involved in focal adhesion, tight junction, and RNA regulation, in addition to TLR pathway and other innate sensing genes. Further investigation using human A549 cells knocked down for various TLR pathway adaptors, revealed significant impacts on the Ad initiation of NF-kB and interferon responses, thus confirming TLR involvement in Ad-mediated immunity across diverse species.