Abstract: Monodispersed polysilsesquioxane (PSQ) spheres with diameters from hundreds of nanometers to several microns have been successfully synthesized; however, the knowledge of their formation mechanism still lags behind. Herein, with methyltrimethoxysilane and 3-mercaptopropyl trimethoxysilane as model silicon sources, the formation process of PSQ spheres in the one-step sol–gel method was revealed for the first time by monitoring the time evolution of particle morphology, size, and size distribution via transmission electron microscopy and dynamic light scattering. A four-stage formation mechanism was proposed: rapid hydrolysis of organic silicon source and subsequent oligomer micelle nucleation, fast growing of nuclei particles and formation of their aggregates, followed by a further relatively fast growth of dispersed particles, and finally a slow growth to form monodispersed PSQ spheres. Due to the reversibility of hydrolysis and condensation reactions, thermodynamically unstable particles gradually transformed to hydrolytic monomers/oligomers and then regrew on the thermodynamically stable particles until the concentration of hydrolytic oligomers reached the dissolution equilibrium in the alkaline reaction solution. The variation of growth rate during the formation process and the effects of NH₄OH concentration on the yield and particle size were investigated to facilitate analyses and understanding of the formation mechanism.