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 IRF3 and IRF7 and partially required the TIR-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 response. This article is protected by copyright. All rights reserved.