Murine norovirus (MNV) is a non-enveloped virus with a positive-sense RNA genome and causes lethal infection in mice. MNV has been used as a model virus for human norovirus (NV) whose in vitro cell culture system has not been available to date since MNV and NV are genetically related. In this study, the genome replication of MNV was investigated using strand-specific RT-PCR in RAW264.7 cells. Reverse transcription (RT) using a sense primer followed by PCR showed that negative-sense RNAs were first detected in RAW264.7 cells between 6 and 9 [3 and 6] hours post infection (h.p.i.). However, these negative-sense RNAs were not detected when cells were treated with a translation inhibitor cycloheximide. Then, RT with an antisense primer followed by PCR was performed to detect positive-sense RNAs. RT-PCR results revealed that the amount of positive-sense RNAs began to increase from 9 [6] h.p.i., indicating the accumulation of the newly synthesized (+)RNA genome. Furthermore, cycloheximide abrogated the increase of newly made RNAs during MNV infection. In conclusion, strand-specific RT-PCR using a sense or antisense primer, in combination with cycloheximide treatment, enabled us to detect positive-sense and negative-sense RNAs selectively and provided a useful tool to understand the replication cycle of MNV.