Akhmeta virus is a zoonotic Orthopoxvirus first identified in 2013 in the country of Georgia. Subsequent ecological investigations in Georgia have found evidence that this virus is widespread in its geographic distribution within the country and in its host-range, with rodents likely involved in its circulation in the wild. Yet, little is known about the pathogenicity of this virus in rodents. We conducted the first laboratory infection of Akhmeta virus in CAST/EiJ Mus musculus to further characterize this novel virus. We found a dose-dependent effect on mortality and weight loss (p < 0.05). Anti-orthopoxvirus antibodies were detected in the second- and third-highest dose groups (5 × 104 pfu and 3 × 102 pfu) at euthanasia by day 10, and day 14 post-infection, respectively. Anti-orthopoxvirus antibodies were not detected in the highest dose group (3 × 106 pfu), which were euthanized at day 7 post-infection and had high viral load in tissues, suggesting they succumbed to disease prior to mounting an effective immune response. In order of highest burden, viable virus was detected in the nostril, lung, tail, liver and spleen. All individuals tested in the highest dose groups were DNAemic. Akhmeta virus was highly pathogenic in CAST/EiJ Mus musculus, causing 100% mortality when ≥3 × 102 pfu was administered.