Rabbit hemorrhagic disease virus (RHDV) is a highly contagious calicivirus that causes peracute hemorrhagic fever and frequently kills rabbits before an effective adaptive immune response can be developed. In Australia and New Zealand, RHDV is employed to manage wild European rabbit (Oryctolagus cuniculus) populations. Although there is no evidence that RHDV replicates in animals other than lagomorphs, the detection of RHDV-specific antibodies and RHDV RNA in mice and other species has raised concerns about the host specificity of the virus. To investigate the replication potential of RHDV in mice (Mus musculus), standard laboratory mice and knockout animals that lack a functional interferon type I receptor were challenged with high doses of RHDV. None of the animals developed clinical signs of illness, and temporal quantification of the viral RNA by real-time PCR did not reveal signs of virus amplification. These data suggest that RHDV cannot replicate in mice—not even in animals with a severely compromised innate immune system.