Interferon regulatory factor 3 (IRF-3) plays an important role in virus and double-stranded RNA-mediated induction of type I interferon and RANTES, DNA damage signaling, tumor suppression, and virus-induced apoptosis. However, cis elements or trans factors responsible for regulating IRF-3 expression remain largely unknown. Here we report that the transcription factor E2F1 negatively regulates the basal transcriptional activity of IRF-3 and deregulates IRF-3 expression at mRNA level. By transient transfection analysis, we demonstrate that the mutation of E2F-binding site results in a profound promotion of IRF-3 promoter activity. Overexpression of E2F1, but not a mutant E2F1, represses the IRF-3 promoter activity in reporter gene assays while knocking down of endogenous E2F1 by shRNA strategy results in enhanced IRF-3 promoter activity. Electrophoretic gel mobility shift assays and antibody competition assays confirm that E2F1 protein binds to the E2F consensus binding site in the IRF-3 promoter. Chromatin immunoprecipitation assays demonstrate that E2F1 interacts with the IRF-3 promoter in vivo. These results suggest that E2F1 negatively regulates IRF-3 transcription through binding to the E2F consensus binding site.