Objective To investigate the natural infection of Theiler's murine encephalomyelitis virus (TMEV) in mice, and to survey the distribution of virus in tissues and the changes of serum antibody in the experimentally TMEV-infected mice. Methods Enzyme linked immunosorbent assay (ELISA) and fluorescence quantitative RT-PCR (qRT-PCR) assay were used to detect the antibody and nucleic acid of TMEV in clinical samples. These samples included SPF mice collected from Guangdong area in 2010-2015, mice obtained from a non-barrier laboratory rodent colony, and wild Rattus norvegicus live-trapped around the non-barrier laboratory rodent colony. 36 ICR mice were intracerebrally inoculated with TMEV BeAn strain. The clinical signs of the animals were observed daily post-inoculation. Three mice were euthanatized at day 0, 3, 7, 10, 17, 21, 31, 39 and 46 post-inoculation (dpi), respectively. Tissue and serum samples were collected for TMEV detection. Results The TMEV antibody and nucleic acid positive rates of SPF mice collected from Guangdong area in 2010-2015 were 5.29% (n=2834) and 27.27% (n=457), respectively. The TMEV antibody and nucleic acid positive rates of the mice obtained from a non-barrier laboratory rodent colony were 71.95% (n=82) and 53.66% (n=82), respectively. The TMEV nucleic acid positive rate of wild Rattus norvegicus was 25.93% (n=27). In the TMEV positive mice, only two mice showed obvious clinical symptoms. The cecal contents, feces and brain samples were the best candidates for qRT-PCR assay. The viral nucleic acid could be detected in the brain, heart, liver, lung and stomach of ICR mice at 3 dpi, but no viral nucleic acid was detected in the spleen, kidney, and cecum. The viruses in liver, heart, lungs and stomach were completely cleared at 10 dpi, and the viruses persisted in the brain throughout the experiment. The TMEV antibody could be detected at 7 dpi, and then the antibody positive rate reached 100% at 17 dpi. The antibody level increased gradually and maintained up to 46 days. ICR mice showed latent infection after TMEV inoculation, with no obvious symptoms and eye pathological changes. Conclusions The experimental mice and wild Rattus norvegicus in Guangdong area are both infected with TMEV, and the infection rate is high. The mice inoculated with TMEV BeAn strain show latent infection. The TMEV antibody produced in mice can be detected at 7 dpi and persisted until the end of the experiment. The viruses are found in the liver, heart, lung and stomach for a short time, but are persisted in the brain for a long time. There is a good consistency of TMEV detection between qRT-PCR and ELISA. The qRT-PCR assay can be used as a powerful complement method for the national standard of laboratory animals.