Objective To investigate the natural infection status of murine norovirus (MNV) in laboratory mice in Shanghai area and isolate MNV from mouse cecal feces. Methods To collect cecal contents and serum samples from 319 specific pathogen-free (SPF) mice coming from different research institutions. Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) were used to detect MNV infection in the mice, respectively. The positive stool samples were diluted and filtered through 0.22 μm membrane, inoculated into RAW 264.7 cells, and then identified by RT-PCR. Results There were 95 positive results in the 319 cecal samples by RT-PCR, and the positive rate was 29.78%. Among 180 serum samples which were tested by RT-PCR, 70 samples were positive by ELISA, and the positive rate was 38.89%. The infected RAW 264.7 cells showed cytopathic effect (CPE) within 72 h. After 3 times of freezing and thawing, RT-PCR obtained a 187 bp band. Conclusions The results from the present study show that there is a high natural infection rate of MNV in laboratory mice in Shanghai area, and the strict breeding management must be strengthened.