This work evaluates the cytotoxicity of spherical mesoporous silica (MS) nano- and microparticles and investigates the effects of particle size, concentration, biodegradation products of MS, residual surfactant, and surfactant removal by extraction and calcination on the cytotoxicity. The results from the intracellular localization of spherical MS nano- and microparticles with different sizes reveal the mechanism for their cytotoxicity and that the smaller particles in nanoscale are more easily endocytosed and consequently located within lysosomes.