The recent discovery demonstrating that the leakage of cathepsin B from mitotic lysosomes assists mitotic chromosome segregation indicates that lysosomal membrane integrity can be spatiotemporally regulated. Unlike many other organelles, structural and functional alterations of lysosomes during mitosis remain, however, largely uncharted. Here, we demonstrate substantial differences in lysosomal proteome, lipidome, size, and pH between lysosomes that were isolated from human U2OS osteosarcoma cells either in mitosis or in interphase. The combination of pharmacological synchronization and mitotic shake-off yielded ~68% of cells in mitosis allowing us to investigate mitosis-specific lysosomal changes by comparing cell populations that were highly enriched in mitotic cells to those mainly in the G1 or G2 phases of the cell cycle. Mitotic cells had significantly reduced levels of lysosomal-associated membrane protein (LAMP) 1 and the active forms of lysosomal cathepsin B protease. Similar trends were observed in levels of acid sphingomyelinase and most other lysosomal proteins that were studied. The altered protein content was accompanied by increases in the size and pH of LAMP2-positive vesicles. Moreover, mass spectrometry-based shotgun lipidomics of purified lysosomes revealed elevated levels of sphingolipids, especially sphin-gomyelin and hexocylceramide, and lysoglyserophospholipids in mitotic lysosomes. Interestingly, LAMPs and acid sphingomyelinase have been reported to stabilize lysosomal membranes, whereas sphingomyelin and lysoglyserophospholipids have an opposite effect. Thus, the observed lysosomal changes during the cell cycle may partially explain the reduced lysosomal membrane integrity in mitotic cells.