Adenovirus (AdV) infection has been rarely documented in cats and other felids. Partial sequences of the hexon and fiber genes of a Hungarian feline adenovirus isolate (FeAdV isolate) showed a close relationship to human AdV (HAdV) type C1. Further molecular and biological characterization is reported here. Whole-genome sequencing revealed two silent mutations in the genome of the FeAdV isolate compared to a HAdV-C1 reference strain (at positions 14,096 and 15,082). Competitive antibody binding to the Coxsackie–adenovirus receptor and α_vβ₃ and α_vβ₅ integrin coreceptors inhibited the binding of the FeAdV isolate in different cell lines, but residual infections suggested alternative entry routes. The FeAdV isolate was found to be more sensitive to heat, low pH and detergents, but more resistant to alkaline and free chlorine treatments, as well as to ribavirin, stavudine and cidofovir treatments, than other human AdV types. We observed a suppression of IL-10 and TGF-β1 production during the entire course of viral replication. This immunomodulation may restore intratumoral immunity; thus, the FeAdV isolate could serve as an alternative oncolytic vector. Collectively, our results support that the Hungarian FeAdV isolate is a variant of common HAdV-C1. The cohabitation of cats with humans might result in reverse zoonotic infection. Felids appear to be susceptible to persistent and productive adenovirus infection, but further studies are needed to better understand the clinical and epidemiological implications.