Several vaccine candidates for Rift Valley Fever (RVF) are in development for use in humans. A promising candidate, ChAdOx1 RVF vaccine, has been developed for use in both humans and animals, and has undergone field trials in livestock in Kenya. We conducted a qualitative study to explore the acceptability of this novel One Health vaccine for Rift Valley Fever prior to phase II/III trials, in two rural Ugandan cohorts between January to June 2020. Data was obtained from 96 semi-structured interviews at Bwindi Impenetrable National Park (BINP) and Kyamulibwa, Kalungu District, in Southern Uganda. The study found that 42% of those interviewed were willing to receive a vaccine that was the same for both humans and animals. 45% of those interviewed said that they would not be willing to receive a One Health vaccine and a further 13% were unsure whether or not they would be happy to receive such a vaccine. Semi-structured interviews were conducted to explore their reasons for and against the acceptability of a novel One Health vaccine to highlight potential barriers to deployment once a vaccine candidate for RVF becomes available.