ObjectiveTo isolate simian adenovirus (SAdV) in vitro. MethodsSimian adenovirus in fecal specimens of monkeys were detected by PCR. SAdV-positive samples were inoculated into BSC-1 cells. The cells were blindly passaged for three times to observe cytopathic effect in cell cultures. The presence of SAdV was confirmed by PCR-sequencing and electron microscopy. ResultsA strain of monkey adenovirus was isolated form Macaca fascicularis in Guangdong province, hence it was named as GD strain. The cytopathic effect was obvious 24 hours after infection. Viral particle-like adenovirus were observed by electron microscopy. The GD strain had high nucleotide sequence homology with HAdV-G. Pol and hexon genes of the isolated strain had 96% and 83% nucleotide sequence homology with SAdV-1, respectively. ConclusionsThe successful isolation of SAdV can facilitate the research for its biology, pathogenesis and laboratory diagnostics, and make it possible to construct SAdV gene transfer vector that benefits development of alternative carrier.