Experimental studies were conducted to determine if hematophagous Diptera were capable of mechanical transmission of Rift Valley fever (RVF) virus to laboratory animals. All species tested (Glossina morsitans, Aedes aegypti, Aedes taeniorhynchus, Culex pipiens, Stomoxys calcitrans, Lutzomyia longipalpis, and Culicoides variipennis) mechanically transmitted the virus to hamsters. Mechanical transmission rates for G. morsitans ranged from 0–100%, with the probability of mechanical transmission positively correlated with initial viremia titer and negatively correlated with the time after virus exposure. Mechanical transmission of RVF virus to lambs was demonstrated with both G. morsitans and Cx. pipiens. These findings demonstrated that mechanical transmission of RVF virus by hematophagous flies may contribute to the natural transmission and dissemination of this virus.