Smallpox preparedness research has led to development of antiviral therapies for treatment of serious orthopoxvirus infections. Monkeypox virus is an emerging, zoonotic orthopoxvirus which can cause severe and transmissible disease in humans, generating concerns for public health. Monkeypox virus infection results in a systemic, febrile-rash illness closely resembling smallpox. Currently, there are no small-molecule antiviral therapeutics approved to treat orthopoxvirus infections of humans. The prairie dog, using monkeypox virus as a challenge virus, has provided a valuable nonhuman animal model in which monkeypox virus infection closely resembles human systemic orthopoxvirus illness. Here, we assess the efficacy of the antiorthopoxvirus compound ST-246 in prairie dogs against a monkeypox virus challenge of 65 times the 50% lethal dose (LD(50)). Animals were infected intranasally and administered ST-246 for 14 days, beginning on days 0, 3, or after rash onset. Swab and blood samples were collected every 2 days and analyzed for presence of viral DNA by real-time PCR and for viable virus by tissue culture. Seventy-five percent of infected animals that received vehicle alone succumbed to infection. One hundred percent of animals that received ST-246 survived challenge, and animals that received treatment before symptom onset remained largely asymptomatic. Viable virus and viral DNA were undetected or at greatly reduced levels in animals that began treatment on 0 or 3 days postinfection, compared to control animals or animals treated post-rash onset. Animals treated after rash onset manifested illness, but all recovered. Our results indicate that ST-246 can be used therapeutically, following onset of rash illness, to treat systemic orthopoxvirus infections.