Ebola virus (EBOV) L polymerase performs de novo replication which is controlled by the specific 3’ leader sequence of EBOV genome. Structural analysis revealed that the 3’ leader RNA binds in the template entry channel with a distinctive stable bend conformation, which is required for the de novo replication activity. These findings have provided a new mechanistic understanding of RNA synthesis for the non-segmented negative-strand RNA virus (nsNSV) including EBOV and revealed important targets for the development of antiviral drugs.