Type I interferons (IFNs) are consequential cytokines in antibacterial defense. Whether and how bacterial pathogens inhibit innate immune receptor-driven type I IFN expression remains mostly unknown. By screening a library of enterohemorrhagic Escherichia coli (EHEC) mutants, we uncovered EhaF, an uncharacterized protein, as an inhibitor of innate immune responses including IFNs. Further analyses identified EhaF as a secreted autotransporter-a type of bacterial secretion system with no known innate immune-modulatory function-that translocates into host cell cytosol and inhibit IFN response to EHEC. Mechanistically, EhaF interacts with and inhibits the MiT/TFE family transcription factor TFE3 resulting in impaired TANK phosphorylation and consequently, reduced IRF3 activation and type I IFN expression. Notably, EhaF-mediated innate immune suppression promotes EHEC colonization and pathogenesis in vivo. Overall, this study has uncovered a previously unknown autotransporter-based bacterial strategy that targets a specific transcription factor to subvert innate host defense.