Rattle-type Fe3O4@SiO2 hollow mesoporous spheres with different particle sizes, different mesoporous shell thicknesses, and different levels of Fe3O4 content are prepared by using carbon spheres as templates. The effects of particle size and concentration of Fe3O4@SiO2 hollow mesoporous spheres on cell uptake and their in vitro cytotoxicity to HeLa cells are evaluated. The spheres exhibit relatively fast cell uptake. Concentrations of up to 150 µg mL−1 show no cytotoxicity, whereas a concentration of 200 µg mL−1 shows a small amount of cytotoxicity after 48 h of incubation. Doxorubicin hydrochloride (DOX), an anticancer drug, is loaded into the Fe3O4@SiO2 hollow mesoporous spheres, and the DOX-loaded spheres exhibit a somewhat higher cytotoxicity than free DOX. These results indicate the potential of Fe3O4@SiO2 hollow mesoporous spheres for drug loading and delivery into cancer cells to induce cell death.