Human-derived replication-defective adenovirus (rAd) vectors have been successfully used to generate protective immunity against acute lethal Ebola virus (EBOV; previously known as Zaire ebolavirus) challenge in rhesus macaques, but their use is limited by pre-existing vector immunity. Here, Stanley et al. describe the development of a vaccine comprised of a chimpanzee-derived rAd (ChAd3) that encodes EBOV glycoprotein. Rhesus macaques administered a single dose of the vaccine were protected from an acute EBOV challenge 5 weeks later, and when boosted with a second vaccine — modified vaccinia Ankara expressing EBOV glycoprotein — were still protected at 10 months.