Rubicon is a part of a Beclin-1-Vps34-containing autophagy complex. Rubicon induces antimicrobial responses upon TLR stimulation, and functions as a feedback inhibitor to prevent unbalanced proinflammatory responses depending on dectin-1 signaling. However, the role played by Rubicon during antiviral immune responses, particularly the type I interferon responses, remains largely unknown. Here, we report that Rubicon acts as a negative regulator for virus-triggered IFN signaling. Knockdown of Rubicon promoted type I interferon signaling and inhibited virus replication, while overexpression of Rubicon has the opposite effect. Rubicon specifically interacts with the IRF association domain (IAD) of IRF3, and this interaction leads to inhibition of the dimerization of IRF3, which negatively regulates IFN-mediated antiviral response. Thus, our findings suggest that the novel additional role of Rubicon as a negative regulator that inhibits the IFN signaling and cellular antiviral responses, providing a novel cellular mechanism of IRF3 inhibition. [The National Research Foundation of Korea (Grant no. 2015020957)]