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 important insights into lipid signaling in innate immune responses. We found that the transfection reagent Lipofectamine can induce type I interferon signaling in macrophages.