Rift Valley fever virus (RVFV), a mosquito-borne virus in the Bunyaviridae family, causes recurrent outbreaks with severe disease in ruminants and occasionally humans. The virus comprises a segmented genome consisting of a small (S), medium (M) and large (L) RNA segment of negative polarity. The M-segment encodes a glycoprotein precursor (GPC) protein that is co-translationally cleaved into Gn and Gc, which are required for virus entry and fusion. Recently we developed a four-segmented RVFV (RVFV-4s) by splitting the M-genome segment, and used this virus to study RVFV genome packaging. Here we evaluated the potential of a RVFV-4s variant lacking the NSs gene (4s-ΔNSs) to induce protective immunity in sheep. Groups of seven lambs were either mock-vaccinated or vaccinated with 10(5) or 10(6) tissue culture infective dose (TCID50) of 4s-ΔNSs via the intramuscular (IM) or subcutaneous (SC) route. Three weeks post-vaccination all lambs were challenged with wild-type RVFV. Mock-vaccinated lambs developed high fever and high viremia within 2 days post-challenge and three animals eventually succumbed to the infection. In contrast, none of the 4s-ΔNSs vaccinated animals developed clinical signs during the course of the experiment. Vaccination with 10(5) TCID50 via the IM route provided sterile immunity, whereas a 10(6) dose was required to induce sterile immunity via SC vaccination. Protection was strongly correlated with the presence of RVFV neutralizing antibodies. This study shows that 4s-ΔNSs is able to induce sterile immunity in the natural target species after a single vaccination, preferably administrated via the IM route.