Liposomes are vesicular structures consisting of an aqueous core surrounded by a lipid bilayer. Apart from the cytosol and lysosomes, no other intracellular compartment has been successfully targeted using liposomal delivery. Here, we report the development of liposomes capable of specific targeting to the endoplasmic reticulum (ER) and associated membranes. Using competition and inhibitor assays along with confocal microscopy, we have determined that ER liposomes utilize scavenger and low‐density lipoprotein receptors for endocytosis and enter cells through a caveolin‐ and microtubule‐dependent mechanism. They traffic intact to the ER, where fusion with the ER membrane occurs after 22–25 min, which was confirmed by fluorescence‐dequenching assays. Once inside the ER, tagged lipids intercalate with the ER membrane and are subsequently incorporated into ER‐assembling entities, such as the ER‐budding viruses hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV), lipid droplets, and secreted lipoproteins. ER liposomes are superior to cytosolic liposome formulations for the intracellular delivery of aqueous cargo, such as HIV‐1 antivirals, and are especially suited for the prolonged delivery of lipids and lipophilic drugs into human cells.—Pollock, S., Antrobus, R, Newton, L., Kampa, B., Rossa, J., Latham, S., Branza Nichita, N., Dwek, R A., Zitzmann, N. Uptake and trafficking of liposomes to the endoplasmic reticulum. FASEB J. 24, 1866–1878 (2010). www.fasebj.org