Ebola virus (EBOV) is a highly infectious and lethal pathogen responsible for sporadic self-limiting clusters of Ebola virus disease (EVD) in Central Africa capable of reaching epidemic status. 100% protection from lethal EBOV-Zaire in Balb/c mice was achieved by rintatolimod (Ampligen) at the well tolerated human clinical dose of 6 mg/kg. The data indicate that the mechanism of action is rintatolimod's dual ability to act as both a competitive decoy for the IID domain of VP35 blocking viral dsRNA sequestration and as a pathogen-associated molecular pattern (PAMP) restricted agonist for direct TLR3 activation but lacking RIG-1-like cytosolic helicase agonist properties. These data show promise for rintatolimod as a prophylactic therapy against human Ebola outbreaks. • Rintatolimod offers 100% protection against Ebola virus in a 100% lethal mouse model. • Protection from in mice is at easily achievable human IV doses of 6 mg/kg. • Rintatolimod is generally well-tolerated in humans at 6 mg/kg. • Rintatolimod blocks EBOV VP35 viral dsRNA binding and is not a RIG-1-like agonist. • Rintatolimod acts as PAMP restricted agonist for direct TLR3 activation. [ABSTRACT FROM AUTHOR]