Alternative human and non-human Ad serotype vectors are currently studied for gene therapy and/or vaccine applications to capitalize upon their likely ability to avoid pre-existing immunity to HAd5. However, relatively little attention has been given to the nature and scope of innate immune responses generated by alternative Ad serotypes. In this study, we characterized several innate immune responses after intravenous administration of wild-type Ad serotypes HAd31, HAd3, HAd5, HAd37, SAd23 and HAd41, representing groups A-F, respectively. Notably, biodistribution studies revealed significant differences between the serotypes, with high levels of HAd3 genomes found in the liver and lung, and HAd37 genomes found in the spleen after systemic administration. Relative to similar treatments with other Ad serotypes, HAd3 and SAd23 induced altered innate immune responses, illustrated by induction of higher levels of cellular gene transcription in several tissues, and higher plasma levels of cytokines and chemokines. We also investigated whether complement interactions have a role in HAd3- and SAd23-induced responses. We confirmed complement dependent gene transcription, plasma cytokine/ chemokine responses, and liver toxicities incurred after administration of HAd3 and SAd23. This study highlights the potential benefits and/or limitations to the proposed use of alternative Ad serotypes for gene therapy or vaccine applications.