Interferon regulatory factor-3 (IRF-3) plays an important role in virus and double-stranded RNA-mediated induction of type I interferon and RANTES (regulated on activation normal T cell expressed and secreted), DNA damage signalling, tumour suppression and virus-induced apoptosis. IRF-3 had recently been shown to contribute to T-cell activation in response to pathogens, which implicated an extensive immunological role for IRF-3. Dendritic cells (DCs) played critical roles as professional APCs in the development of immune responses. However, it was unclear whether IRF-3 had any effect on phenotype or function of DCs. In this study, it was shown that IRF-3 acted as a promoter of DC maturation. The level of IRF-3 expression was transiently upregulated and accumulated in the nucleus in TNF-α-induced immune maturation of mice DC cells. Knockdown of IRF-3 by small interfering RNA in DC cells resulted in both phenotypic and functional immaturation, even without TNF-α treatment. Overall, our data demonstrated for the first time that IRF-3 was a critical regulator of mice DC maturation.