Monkeypox virus (MPXV) is a zoonotic disease endemic in Central and West Africa and is the most virulent orthopoxvirus affecting humans since the eradication of variola virus (VARV). In order to assess their reservoir potential, wild-caught rope squirrels were experimentally infected via intranasal and intradermal exposure with a recombinant MPXV strain from Central Africa engineered to express the luciferase gene. After infection, we monitored viral replication via in vivo bioluminescent imaging and viral shedding via culture and PCR. MPXV infection in African rope squirrels caused mortality and moderate to severe morbidity, with clinical signs including pox lesions in the skin, eyes, mouth and nose, dyspnea, and profuse nasal discharge. Both intranasal and intradermal exposures induced high levels of viremia, fast systemic spread, and long periods of viral shedding. Shedding and luminescence peaked at day 6 post infection and was still detectable after 15 days. Evidence of viral persistence in tissues by real-time PCR was observed in animals that survived infection. Interestingly, one sentinel animal, housed in the same room but in a separate cage, also developed severe MPX disease and was euthanized. This study highlights the epidemiological importance of African rope squirrels and suggests the potential role of this rodent species as a natural reservoir host of MPXV and a source of direct transmission to humans and other animals in endemic MPX regions.