We describe the synthesis of diacyltetrol-based hybrid lipids in which one of the hydroxymethyl groups is modified with an anionic phospholipid headgroup. The hybrid lipids form a monolayer at the air–water interface. In aqueous solution, these lipids form stable liposomes that exhibit a negative surface potential across a wide pH range. The liposomes aggregate in the presence of Ca²⁺ ions and release encapsulated cationic reporter rhodamine 6G (R6G) at a faster rate than anionic reporter carboxyfluorescein (CF). The hybrid lipids strongly interact with the C1b subdomain of the protein kinase C (PKC)-θ isoform. These new lipids structurally mimic diacylglycerol and conventional phospholipids, and provide an opportunity to explore their physicochemical properties.