Enterovirus 71 (EV71), a major pathogen that is responsible for causing hand-foot-and-mouth disease (HFMD) worldwide, is a member of the Human Enterovirus species A, family Picornaviridae. HFMD that is caused by EV71 is usually characterized by vesicular lesions on the skin and oral mucosa and high morbidity rates in children; additionally, occasional fatal cases have been reported involving brainstem encephalitis and myelitis associated with cardiopulmonary collapse. Although viral pathogenesis in humans is unclear, previous animal studies have indicated that EV71, inoculated via various routes, is capable of targeting and injuring the central nervous system (CNS). We report here the pathogenic process of systemic EV71 infection in rhesus monkeys after inoculation via intracerebral, intravenous, respiratory and digestive routes. Infection with EV71 via these routes resulted in different rates of targeting to and injury of the CNS. Intracerebral inoculation resulted in pulmonary edema and hemorrhage, along with impairment of neurons. However, intravenous and respiratory inoculations resulted in a direct infection of the CNS, accompanied by obvious inflammation of lung tissue, as shown by impairment of the alveoli structure and massive cellular infiltration around the terminal bronchioles and small vessels. These pathological changes were associated with a peak of viremia and dynamic viral distribution in organs over time in the infected monkeys. Our results suggest that the rhesus monkey model may be used to study not only the basic pathogenesis of EV71 viral infections, but also to examine clinical features, such as neurological lesions, in the CNS and pathological changes in associated organs.