Well-dispersed mesoporous SiO2 colloidal spheres (MSCSs) were prepared with hexadecyltrimethyl ammonium phosphomolybdate (M-6) as template. The MSCSs were characterized by scanning electron microscopy, transmission electron microscopy, X-ray powder diffraction, and Fourier transform infrared spectroscopy, etc. The diameter and the uniform pore size of the MSCSs are similar to 200 nm and similar to 2.85 nm, respectively. By rational tuning the synthesis conditions such as the concentration of M-6, the volume ratio of water to ethanol, the amount of tetraethoxysilane, etc., the morphology, size and dispersity of the SiO2 products can be easily controlled. The formation mechanism of the as-prepared MSCSs was also proposed. Treated with sulfuric acid, the resulted MSCSs exhibited excellent catalytic activity in the synthesis of biodiesel via the esterification of long-chain free fatty acids. Crown Copyright (C) 2015 Published by Elsevier B.V. All rights reserved.