Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and is also sometimes associated with serious neurological disorders. In this study, we characterized the antigenicity and tissue specificity of an attenuated strain of EV71 [EV71(S1-3')], which belongs to genotype A, in a monkey infection model. Three cynomolgus monkeys were inoculated with EV71(S1-3'), followed by lethal challenge with the parental virulent strain EV71(BrCr-TR) via an intravenous route on day 45 postinoculation of EV71(S1-3'). Monkeys inoculated with EV71(S1-3') showed a mild neurological symptom (tremor) but survived lethal challenge by virulent EV71(BrCr-TR) without exacerbation of the symptom. The immunized monkey sera showed a broad spectrum of neutralizing activity against different genotypes of EV71, including genotypes A, B1, B4, C2, and C4. For the strains examined, the sera showed the highest neutralization activity against the homotype (genotype A) and the lowest neutralization activity against genotype C2. The order of decreasing neutralization activity of sera was as follows: A > B1 > C4 > B4 > C2. To examine the tissue specificity of EV71(S1-3'), two monkeys were intravenously inoculated with EV71(S1-3'), followed by examination of virus distribution in the central nervous system (CNS) and extraneural tissues. In the CNS, EV71(S1-3') was isolated only from the spinal cord. These results indicate that EV71(S1-3') acts as an effective antigen, although this attenuated strain was still neurotropic when inoculated via the intravenous route.