Silver nanoparticles (nanosilver) are broadly used today in textiles, food packaging, household devices and bioapplications, prompting a better understanding of their toxicity and biological interactions. In particular, the cytotoxicity of nanosilver with respect to mammalian cells remains unclear, because such investigations can be biased by the nanosilver coatings and the lack of particle size control. Here, nanosilver of well-defined size (5.7 to 20.4 nm) supported on inert nanostructured silica is produced using flame aerosol technology. The cytotoxicity of the prepared nanosilver with respect to murine macrophages is assessed in vitro because these cells are among the first to confront nanosilver upon its intake by mammals. The silica support facilitates the dispersion and stabilization of the prepared nanosilver in biological suspensions, and no other coating or functionalization is applied that could interfere with the biointeractions of nanosilver. Detailed characterization of the particles by X-ray diffraction and electron microscopy reveals that the size of the nanosilver is well controlled. Smaller nanosilver particles release or leach larger fractions of their mass as Ag+ ions upon dispersion in water. This strongly influences the cytotoxicity of the nanosilver when incubated with murine macrophages. The size of the nanosilver dictates its mode of cytotoxicity (Ag+ ion-specific and/or particle-specific). The toxicity of small nanosilver (<10 nm) is mostly mediated by the released Ag+ ions. The influence of such ions on the toxicity of nanosilver decreases with increasing nanosilver size (>10 nm). Direct silver nanoparticle–macrophage interactions dominate the nanosilver toxicity at sizes larger than 10 nm.