Interferon regulatory factor 3 (IRF3) is an essential transcriptional regulator of the interferon genes. IRF3 is constitutively present in a latent conformation in the cell cytoplasm. In cells infected by Sendai virus, IRF3 becomes phosphorylated, homodimerizes, translocates to the nucleus, binds to target genes and activates transcription by interacting with CBP/p300 co-activators. In this study, we report that in non-infected cells IRF3 is post-translationally modified by S-glutathionylation. Upon viral-infection, it undergoes a deglutathionylation step that is controlled by the cytoplasmic enzyme glutaredoxin-1 (GRX-1). In virus-infected GRX-1 knockdown cells, phosphorylation, homodimerization and nuclear translocation of IRF3 were not affected, but the transcriptional activity of IRF3 and the expression of interferon-beta (IFNbeta), were severely reduced. We show that deglutathionylation of IRF3 is necessary for efficient interaction of IRF3 with CBP, an event essential for transcriptional activation of the interferon genes. Taken together, these findings reveal a crucial role for S-glutathionylation and GRX-1 in controlling the activation of IRF3 and IFNbeta gene expression.