Eggert and colleagues report the first comprehensive analysis of how the lipidome changes from interphase to cytokinesis. They identified 11 lipid species (out of thousands) that accumulated fourfold in dividing cells, including sphingolipids and ceramides. Further experiments revealed that films made from mixtures of lipids from interphase or dividing cells had different physical properties, suggesting that lipids may have a mechanical role in cell division. RNAi-mediated knockdown of 23 lipid biosynthetic enzymes caused cytokinetic failure. Moreover, some of the lipids that were upregulated in dividing cells accumulated in midbodies (all sphingolipid derivatives), which indicates that local changes at the plasma membrane may have crucial functions during cell division, either directly, or by affecting membrane-associated signalling and the cytoskeleton. This study opens new avenues for investigating the role of specific lipids at the subcellular level.