We aimed to study infections in neonatal ICR mice of different ages infected with Enterovirus 71(EV-A71)through three routes of infection, and to explore the dynamic distribution and infection mechanism of EV-A71 in vivo.Three-,5-and 9-day-old neonatal ICR mice were infected with an EV-A71 strain isolated from a child with severe hand, foot and mouth disease through intramuscular(IM), intraperitoneal (IP)and intracerebral (IC)injection, respectively. Consequently, blood, brain, hind-limb muscle, heart, and intestines of mice were collected at regular intervals. Changes in viral load in organs were measured using real-time polymerase chain reaction. Hematoxylin and eosin staining and immunohistochemical (IHC)analyses were undertaken to detect pathogenic and pathologic changes in infected mice.Five-day-old neonatal mice infected with EV-A71 through IM,IP or IC routes had obvious neurologic symptoms and a high mortality rate. Symptoms were alleviated slightly with increasing age of mice upon injection. However, the pathogenicity associated with IM and IP injections was more severe than that of IC injection. Also, the mortality rates of IM and IP injections were significantly higher than that of IC injection. Compared with the control group, the mean body weight(in g)of 3-day-old neonatal mice at 6days post-infection(dpi)injected by IM,IP and IC routes decreased by 1.54(31.43%),1.31(15.06%)and 2.52(44.28%),respectively. Similarly, the mean body weight(in g)of 5-day-old neonatal mice at 6dpi injected by IM and IP decreased by 0.605(8.95%),0.886(15.51%),whereas that of mice injected by IC increased by 0.904(14.70%).The body weight of all infection groups was significantly lower than that of the control group(P<0.05).All 3-day-old neonatal mice infected with EV-A71 through IM,IP and IC routes died at 9dpi.Survival rates of 5-day-old neonatal mice infected through IM,IP and IC routes at 9dpi and14 dpi were 42.8%,25%,and 87.5%,and 0%,0%,and 25%,respectively.Those of 9-day-old neonatal mice at 14 dpi were 70%,69.23% and 100%,respectively.Pathologic and IHC examination showed that EV-A71 had a strong preference for infecting nervous systems and skeletal muscle, and could also lead to: viremia; necrosis of brain neurons and skeletal muscle; myocardial interstitial edema; inflammatory response of multiple organs. These data suggest that 5-day-old ICR neonatal mice injected through IM or IP routes can establish an ideal model of infection by EV-A71 in mice.