ABSTRACT Flaviviruses are important human pathogens including viruses such as West Nile, dengue, and Zika. No specific treatments or vaccines are available against these infections, and no biomarkers exist that allow predicting their outcome, characterized by important clinical variability. Understanding innate antiviral immune responses against flaviviruses, could lead to the identification of disease susceptibility biomarkers and therapeutic targets. We recently demonstrated that the type I interferon-stimulated protein Schlafen 11 (SLFN11) is a novel and potent flavivirus restriction factor. In this application we aim to define, at the molecular level, several aspects of the mechanism of action of SLFN11 and determine the anti-flavivirus activity of other members of the SLFN family, as well as the in vivo relevance of this defense system. Therefore, this investigation is highly relevant and will significantly advance our understanding of the antiviral mechanism of action of SLFN proteins, which is currently unknown.