Vectors based on nonhuman adenoviruses (Ads) such as porcine Ad serotype 3 (PAd3) and bovine Ad serotype 3 (BAd3) are being developed as alternative to human adenoviral (HAd) vectors to circumvent the problem of preexisting immunity in humans. To assess the usefulness of PAd3 and BAd3 vectors for in vivo gene delivery, we examined and compared the vector biodistribution, persistence, state of vector genome, efficiency of transgene and vector genes expression, induction of innate immunity and role of Kupffer cells (KCs) by replication defective BAd3, PAd3 and HAd serotype 5 (HAd5) vectors in a mouse model following intravenous inoculation. Immunological cross-reactivity among HAd5, BAd3 and PAd3 at the humoral and cellular levels was also evaluated. BAd3 vector efficiently transduced the heart, kidney and lung, in addition to the liver and spleen and persisted for a longer duration compared to the PAd3 or HAd5 vector. Biodistribution of the PAd3 vector was comparable to that of the HAd5 vector but showed more rapid vector clearance. In contrast to PAd3 and HAd5 vectors, the ability of BAd3 vector to evade uptake by KCs, can partially explain the prolonged persistence of BAd3 vector at higher levels in most tissues. Only linear episomal-form of BAd3, PAd3, or HAd5 vector genomes was detected. All three vectors efficiently expressed the green fluorescent protein (GFP) transgene proportionate to vector levels in various tissues. Furthermore, leaky expression of vector genes, both the early (E4) and the late (hexon) was observed by all three vectors and gradually declined with time. BAd3 and PAd3 vectors were more potent inducer of variety of cytokines and chemokines production and TLRs expression. No significant immunological cross-reactivity among HAd5, BAd3 and PAd3 was observed. These results highlight the potential of BAd3 and PAd3 vectors as alternative or supplement to HAd5 for gene delivery applications.