A licensed vaccine against Ebola virus (EBOV) remains unavailable, despite >11 000 deaths from the 2014–2016 outbreak of EBOV disease in West Africa. Past studies have shown that recombinant vaccine viruses expressing EBOV glycoprotein (GP) are able to protect nonhuman primates (NHPs) from a lethal EBOV challenge. However, these vaccines express the viral GP–based EBOV variants found in Central Africa, which has 97.3% amino acid homology to the Makona variant found in West Africa. Our previous study showed that a recombinant adenovirus serotype 5 (Ad5)–vectored vaccine expressing the Makona EBOV GP (MakGP) was safe and immunogenic during clinical trials in China, but it is unknown whether the vaccine protects against EBOV infection. Here, we demonstrate that guinea pigs immunized with Ad5-MakGP developed robust humoral responses and were protected against exposure to guinea pig–adapted EBOV. Ad5-MakGP also elicited specific B- and T-cell immunity in NHPs and conferred 100% protection when animals were challenged 4 weeks after immunization. These results support further clinical development of this candidate and highlight the utility of Ad5-MakGP as a prophylactic measure in future outbreaks of EBOV disease.