Spherical monodispersed bioactive particles are potential candidates for nanocomposite synthesis or as injectable particles that could be internalized by cells for the local sustained delivery of inorganic therapeutic ions (e.g., calcium or strontium). Particles are also likely to be released from porous bioactive glass and sol–gel hybrid scaffolds as they degrade; thus, it is vital to investigate their interaction with cells. Spherical monodispersed bioactive glass particles (mono-SMBG), with diameters of 215 ± 20 nm are synthesized using a modified Stöber process. Confocal and transmission electron microscopy demonstrate that mono-SMBGs are internalized by human bone marrow (MSCs) and adipose-derived stem cells (ADSCs) and located within cell vesicles and in the cytoplasm. Particle dissolution inside the cells is observed. Alamar Blue, MTT and Cyquant assays demonstrate that 50 μg mL−1 of mono-SMBGs did not inhibit significantly MSC or ADSC metabolic activity. However, at higher concentrations (100 and 200 μg mL−1) small decrease in metabolic activity and total DNA is observed. Mono-SMBG did not induce ALPase activity, an early marker of osteogenic differentiation, without osteogenic supplements; however, in their presence osteogenic differentiation is achieved. Additionally, large numbers of particles are internalized by the cells but have little effect on cell behavior.