Objective To evaluate the feasibility of endotracheal intubation route in preparation of H5N1 virus infection in rhesus macaques and to obtain a more sensitive animal model． Methods The rhesus macaques were infected with H5N1 avian influenza virus by endotracheal intubation． Clinical signs were assessed daily，and throat swab，nasal wash，major organs and blood were collected for detection of virus load，isolation and blood routine analysis． Results After infection the animals exhibited anorexia，lethargy and transient fever，and the total number of circulating leukocytes and lymphocytes was decreased． Virus isolation，RT-PCR assay of the throat swabs，nasal washs materials showed that the intestine，lung，brain，spleen，kidney，heart and liver were the target of the virus replication and the virus could replicate in organs outside the lung． In addition，virus was also detected in the blood 3 days after infection． Conclusions Rhesus macaques can be infected with H5N1 virus by endotracheal intubation，and it is proved to be a good method to obtain a more sensitive animal model of H5N1 virus infection． The experimental results will lay the foundation for further studies of pathogenetic mechanism of H5N1 infection．