Preventing filoviruses in the entry stage is an attractive antiviral strategy. Taking aloperine, a Chinese natural herb with an endocyclic skeleton, as the lead, 23 new aloperine derivatives were synthesized and evaluated for their anti-filovirus activities including ebola virus (EBOV) and marburg virus (MARV) using pseudotyped virus model. Structure-activity relationship (SAR) analysis indicated that the introduction of a 12N-dichlorobenzyl group was beneficial for the potency. Compound 2e exhibited the most potent anti-EBOV and anti-MARV effects both in vitro and in vivo. It also displayed a good pharmacokinetic and safety profile in vivo, indicating an ideal druglike feature. The primary mechanism study showed that 2e could block a late stage of viral entry, mainly through inhibiting cysteine cathepsin B activity of host components. We consider compound 2e to be a promising broad-spectrum anti-filovirus agent with the advantages of a unique chemical scaffold and a specific biological mechanism. Aloperine derivative 2e was identified to exert a broad-spectrum anti-filovirus activity mainly through targeting a host protein cysteine cathepsin B to block a late stage of viral entry. Display omitted •23 new aloperines were synthesized and evaluated for the anti-EBOV activity.•Compound 2e exhibited potent anti-EBOV/MARV effects both in vitro and in vivo.•Compound 2e displayed a good pharmacokinetic and safety profile in vivo.•Compound 2e could inhibit cat B activity to block the late stage of viral entry.