This protocol describes a novel method that enables transfection of plasmids and siRNAs into the mouse intestinal epithelium. The mouse was anesthetized with isoflurane, and the small intestine was pulled out from the peritoneal cavity. The small intestinal lumen was then washed with buffer containing a reducing agent, dithiothreitol, to remove mucus, and injected with transfection solution. To achieve efficient gene delivery, we used a hemagglutinating virus of Japan envelope (HVJ-E)-based transfection reagent that can incorporate small molecules, such as proteins, plasmids and siRNAs, and transfer them into cells. To confirm efficient transduction of the incorporated molecules, we transduced fluorescently-labeled molecules, such as Cy3-labeled siRNAs, into the intestinal epithelium by this method, and then observed the tissue sections by fluorescence microscopy. This protocol provides a novel method to analyze function of genes in the intestinal epithelium.