Objective To establish a rhesus monkey model of SIV infection by mucosal route, and compare possible differences in disease course after SIV inoculation in rhesus monkeys by intravenous, intravaginal and intrarectal routes. Methods SIVmac251 strain was used to inoculate three rhesus monkeys by intravenous, intravaginal and intrarectal routes, respectively. Regular sampling, viral load detection, CD4+ cells measurement and virus isolation were approached to identify the results. Results From 7d to 56d (at present) after inoculation, the results of virus isolation, DNA-PCR detection were positive. Viral loads of plasma from S153, S172 and S142 were measured during earlier stage. The antibodies in plasma were detected positively from 14d and up to now. Ratio of CD4+/CD8+ from all monkeys became inverted from 28d after inoculation. Conclusion It has succeeded to inoculate rhesus monkeys with SIVmac251 strain by different routes, and for the first time in China, to be successful in establishment of a SAIDS animal model infected by a mucosal route.