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The commercial transfection reagent Lipofectamine has been widely used for cytoplasmic delivery of nucleic acids and for cytosolic engagement with intracellular innate immune sensors to trigger type I interferon (IFN) production. However, the effect of Lipofectamine alone on type I IFN response has not been studied in detail. Here, we show that Lipofectamine induced type I IFN signaling in both RAW 264.7 macrophage‐like cells and primary bone marrow‐derived macrophages. Type I IFN induction was dependent on interferon regulatory factor (IRF)3 and IRF7 and partially required the toll/interleukin‐1 receptor‐domain‐containing adapter‐inducing interferon‐β. In contrast, the transfection reagent Xfect did not activate type I IFN signaling. Our study highlights the potential confounding experimental interpretation when using Lipofectamine‐based transfection for delivering intracellular ligands and provides …