Non-enzymatic synthesis of amphiphilic diesters from polar diols and fatty acyl donors in water is a fundamental challenge in chemistry. Without a solution, it is unclear how phospholipid formation may have occurred under prebiotic or early biological conditions, since glycerol phosphates (canonical lipid headgroups) resist aqueous lipidation in the absence of enzymes or organic cosolvents.1 Here we demonstrate that, in contrast to glycerols, canonical 5’-ribonucleotides undergo fatty esterification of their diol in water, forming a new class of amphiphiles – phosphonucleolipids. We characterise this reaction and the products, and show that phosphonucleolipid formation directly induces spontaneous self-assembly of stable, cell-like vesicles capable of encapsulating water-soluble molecules. Our results identify nucleotides as privileged substrates for aqueous diol lipidation and demonstrate a spontaneous route to membrane-forming amphiphiles in water, with implications for prebiotic chemistry and the enzyme-free construction of synthetic artificial cells.