Cell surface exposed cholesterol is critical for cell attachment and invasion of many viruses and bacteria, including the bacterium Salmonella, which causes typhoid fever and gastroenteritis. Using flow cytometry and 3D confocal fluorescence microscopy, we found that mitotic cells - even though representing only 1–4% of an exponentially growing population - were much more efficiently targeted for invasion by Salmonella. This targeting was not dependent on the spherical shape of mitotic cells, but was instead SipB- and cholesterol-dependent. Thus, we measured the levels of plasma membrane and cell surface cholesterol along the cell cycle using respectively brief staining with filipin and a fluorescent ester of polyethylene glycol-cholesterol that cannot flip through the plasma membrane, and found that both were maximal during mitosis. This increase was not only due to the rise in global cell cholesterol levels along the cell cycle but also to a transient loss in cholesterol asymmetry at the plasma membrane during mitosis. We measured that cholesterol, but not phosphatidylserine, changed from a ∼20∶80 outer∶inner leaflet repartition during interphase to ∼50∶50 during metaphase, suggesting this was specific to cholesterol and not due to a broad change of lipid asymmetry during metaphase. This explains the increase in outer surface levels that make dividing cells more susceptible to Salmonella invasion and perhaps to other viruses and bacteria entering cells in a cholesterol-dependent manner. The change in cholesterol partitioning also favoured the recruitment of activated ERM (Ezrin, Radixin, Moesin) proteins at the plasma membrane and thus supported mitotic cell rounding.